Overexpression Of ADAR1 Research Articles

Abstract PO1-24-01: ADAR1 drives ribosomal biogenesis by regulating NOP14 and stabilizing FBL-NOP58 complex in breast cancer

Abstract Background Breast cancer (BC) has been widely accepted as a highly heterogeneous disease. Transcriptional and translational dysregulation is commonly found in BC patients. Adenosine-to-inosine (A-to-I) editing on RNA is the most abundant RNA editing form in human, mediated mainly by ADARs. Though the role of ADAR1 in RNA editing and immune response has been deeply investigated, the change of the translational profile and the biology sequalae after base editing are still unclear. In this study, we uncovered a previously unknown role of ADAR1 in the regulation of ribosome biosynthesis and translational dysregulation in BC cells. Method RNA-seq was used for gene set enrichment analysis (GSEA) and A-to-I editing analysis. Transmission electron microscopy (TEM) was conducted to visualize the nucleolar architecture. Nuclear rRNA abundances of 18S and 28S rRNAs were monitored using Qsep100. Ribosomal fractions were isolated with a Beckman SW41Ti rotor and quantified on a 2100 Bioanalyzer. 3' Untranslated region (3′UTR) reporter construction and dual-luciferase reporter assay were used to validate the correlation. In-vitro transcription, RNA pull-down assay and sanger sequencing were used to directly confirm the direct interaction and A-to-I editing status. Cell proliferation, migration assay, apoptosis assay in vitro and xenograft mouse model in vivo were performed for phenotype study. Co-immunoprecipitation (Co-IP) was used to identify the interactive protein. Immunofluorescence (IF) assay was applied to indicate the co-localization. Immunohistochemistry (IHC) staining was applied to tissue microarrays and assess of expression. Survival analysis was made by Kaplan-Meier method. RNA polymerase I inhibitor CX-5461 was used for treatment. Result Through bioinformatic analysis with RNA-seq data from cell lines and clinical samples, we found the expression of ADAR1 was correlated with signatures of ribosomes. Consistently, BC cells with overexpression of ADAR1 showed larger nucleoli and contained more 18S and 28S rRNAs. To uncover the potential ribosomal genes involved in this process, we analyzed the transcriptional profile of 88 BC samples in combination with ADAR1 editing profiles identified by comparing ADAR1-null and control cells. Nucleolar Protein 14 (NOP14), a nucleolar ribosome biogenesis gene, was found to be hyper-edited. Phenotype experiments revealed that NOP14 promotes tumor growth and invasion in vitro and in vivo. The RNA and protein level of NOP14 is regulated by ADAR1 through A-to-I editase activity. Mechanically, ADAR1 binds directly with NOP14 transcripts in the 3’UTR region through Sanger sequencing and RNA-pull down. The binding region of NOP14 with ADAR1 was also a target of miR-939-3p, while the ADAR1 mediated-editing led to less binding of miR-939-3p with NOP14 and resulted in NOP14 up-regulation. Notably, NOP14 facilitates the assembly of the box C/D small nucleolar ribonucleoprotein (snoRNP) and stabilize the interaction of fibrillarin (FBL)-nucleolar Protein 58 (NOP58) with this complex. Loss of NOP14 impaired the nucleolar distribution of FBL and the abundance of ribosomes. CX-5461, an RNA polymerase I inhibitor, suppressed the overexpression of ribosomes in ADAR1 high cells and displayed a strong inhibition efficacy on tumor growth both in vitro and in vivo. Conclusion Taken together, these results shed light on the role of ADAR1 in ribosomal biogenesis in breast cancer. The promising results also provide potential targets in BCs with high ADAR1 by suppressing translational dysregulation. Citation Format: Liren Wangxu, Hengyu Ren, Zhibo Shao, Jiong Wu, Yayun Chi. ADAR1 drives ribosomal biogenesis by regulating NOP14 and stabilizing FBL-NOP58 complex in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-01.

Role of ADAR1 on Proliferation and Differentiation in Porcine Preadipocytes.

Recent research has identified ADAR1 as a participant in the regulation of lipid accumulation in mice. However, there are no reports on the roles of ADAR1 in proliferation, apoptosis and differentiation of porcine preadipocytes. In this study, we investigated the role of ADAR1 in differentiation, proliferation and apoptosis of porcine preadipocytes using CCK-8, EdU staining, cell cycle detection, RT-qPCR, Western blot, a triglyceride assay and Oil Red O staining. The over-expression of ADAR1 significantly promoted proliferation but inhibited the differentiation and apoptosis of porcine preadipocytes. The inhibition of ADAR1 had the opposite effect on the proliferation, differentiation and apoptosis of porcine preadipocytes with over-expressed ADAR1. Then, the regulation mechanisms of ADAR1 on preadipocyte proliferation were identified using RNA-seq, and 197 DEGs in response to ADAR1 knockdown were identified. The MAPK signaling pathway is significantly enriched, indicating its importance in mediating fat accumulation regulated by ADAR1. The study's findings will aid in uncovering the mechanisms that regulate fat accumulation through ADAR1.

A-to-I RNA editing by ADAR and its therapeutic applications: From viral infections to cancer immunotherapy.

ADAR deaminases catalyze adenosine-to-inosine (A-to-I) editing on double-stranded RNA (dsRNA) substrates that regulate an umbrella of biological processes. One of the two catalytically active ADAR enzymes, ADAR1, plays a major role in innate immune responses by suppression of RNA sensing pathways which are orchestrated through the ADAR1-dsRNA-MDA5 axis. Unedited immunogenic dsRNA substrates are potent ligands for the cellular sensor MDA5. Upon activation, MDA5 leads to the induction of interferons and expression of hundreds of interferon-stimulated genes with potent antiviral activity. In this way, ADAR1 acts as a gatekeeper of the RNA sensing pathway by striking a fine balance between innate antiviral responses and prevention of autoimmunity. Reduced editing of immunogenic dsRNA by ADAR1 is strongly linked to the development of common autoimmune and inflammatory diseases. In viral infections, ADAR1 exhibits both antiviral and proviral effects. This is modulated by both editing-dependent and editing-independent functions, such as PKR antagonism. Several A-to-I RNA editing events have been identified in viruses, including in the insidious viral pathogen, SARS-CoV-2 which regulates viral fitness and infectivity, and could play a role in shaping viral evolution. Furthermore, ADAR1 is an attractive target for immuno-oncology therapy. Overexpression of ADAR1 and increased dsRNA editing have been observed in several human cancers. Silencing ADAR1, especially in cancers that are refractory to immune checkpoint inhibitors, is a promising therapeutic strategy for cancer immunotherapy in conjunction with epigenetic therapy. The mechanistic understanding of dsRNA editing by ADAR1 and dsRNA sensing by MDA5 and PKR holds great potential for therapeutic applications. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Disease.

ADAR1 regulates vascular remodeling in hypoxic pulmonary hypertension through N1-methyladenosine modification of circCDK17

Pulmonary hypertension (PH) is an extremely malignant pulmonary vascular disease of unknown etiology. ADAR1 is an RNA editing enzyme that converts adenosine in RNA to inosine, thereby affecting RNA expression. However, the role of ADAR1 in PH development remains unclear. In the present study, we investigated the biological role and molecular mechanism of ADAR1 in PH pulmonary vascular remodeling. Overexpression of ADAR1 aggravated PH progression and promoted the proliferation of pulmonary artery smooth muscle cells (PASMCs). Conversely, inhibition of ADAR1 produced opposite effects. High-throughput whole transcriptome sequencing showed that ADAR1 was an important regulator of circRNAs in PH. CircCDK17 level was significantly lowered in the serum of PH patients. The effects of ADAR1 on cell cycle progression and proliferation were mediated by circCDK17. ADAR1 affects the stability of circCDK17 by mediating A-to-I modification at the A5 and A293 sites of circCDK17 to prevent it from m1A modification. We demonstrate for the first time that ADAR1 contributes to the PH development, at least partially, through m1A modification of circCDK17 and the subsequent PASMCs proliferation. Our study provides a novel therapeutic strategy for treatment of PH and the evidence for circCDK17 as a potential novel marker for the diagnosis of this disease.

ADAR1 Isoforms Regulate Let-7d Processing in Idiopathic Pulmonary Fibrosis.

Double-stranded RNA adenosine deaminase 1 (ADAR1) is significantly down-regulated in fibroblasts derived from Idiopathic Pulmonary Fibrosis (IPF) patients, and its overexpression restored levels of miRNA-21, PELI1, and SPRY2. There are two ADAR1 isoforms in humans, ADAR1-p110 and ADAR1-p150, generated by an alternative promoter. Let-7d is considered an essential microRNA in Pulmonary Fibrosis (PF). In silico analysis revealed COL3A1 and SMAD2, proteins involved in the development of IPF, as Let-7d targets. We analyzed the role of ADAR1-p110 and ADAR1-p150 isoforms in the regulation of Let-7d maturation and the effect of this regulation on the expression of COL3A1 and SMAD2 in IPF fibroblast. We demonstrated that differential expression and subcellular distribution of ADAR1 isoforms in fibroblasts contribute to the up-regulation of pri-miR-Let-7d and down-regulation of mature Let-7d. Induction of overexpression of ADAR1 reestablishes the expression of pri-miR-Let-7d and Let-7d in lung fibroblasts. The reduction of mature Let-7d upregulates the expression of COL3A1 and SMAD2. Thus, ADAR1 isoforms and Let-7d could have a synergistic role in IPF, which is a promising explanation in the mechanisms of fibrosis development, and the regulation of both molecules could be used as a therapeutic approach in IPF.

ADAR1 inhibits adipogenesis and obesity by interacting with Dicer to promote the maturation of miR-155-5P.

Adipogenesis is closely related to various metabolic diseases, such as obesity, type 2 diabetes, cardiovascular diseases and cancer. This cellular process is highly dependent on the expression and sequential activation of a diverse group of transcription factors. Here, we report that ADAR1 (also known as ADAR) could inhibit adipogenesis through binding with Dicer (also known as DICER1), resulting in enhanced production of miR-155-5p, which downregulates the adipogenic early transcription factor C/EBPβ. Consequently, the expression levels of late-stage adipogenic transcription factors (C/EBPα and PPARγ) are reduced and adipogenesis is inhibited. More importantly, in vivo studies reveal that overexpression of ADAR1 suppresses white adipose tissue expansion in high fat diet-induced obese mice, leading to improved metabolic phenotypes, such as insulin sensitivity and glucose tolerance.

Presumptive Evidence for ADAR1 A-to-I Deamination at WA-sites as the Mutagenic Genomic Driver in Hepatocellular and Related ADAR1-Hi Cancers

In hepatocellular cancer (HCC) there is an over expression of the RNA editing enzyme ADAR1. Further, the prominent genomic somatic mutation signature in HCC is almost exclusively focused on mutations at A:T base pairs where A-to-G mutations far exceed T-to-C mutations (when read on the non-transcribed strand). A clear mechanism for this extreme transcriptional strand biased mutation signature, putatively associated with over expression of ADAR1 deaminase, is yet to be explicitly demonstrated. The standard description of this strand bias has been nominally called “Transcription Coupled Damage” (TCD) to distinguish it from more conventional “Transcription Coupled Repair” (TCR). We show that the TCD description does not satisfy all features of the molecular evidence. The conventional view is that ADAR1 is thought to target adenosines at WA-sites for editing to inosine (I) in double stranded RNA stem-loop structures in transcripts. Here we show that the totality of the molecular and cellular data on these mutation signatures provides strong presumptive evidence for a clear role for ADAR1-mediated A-to-I deamination at WA-sites as the mutagenic driver in hepatocellular and possibly other related ADAR1-Hi cancers displaying biased mutation features at A:T base pairs.

Abstract P4-08-03: Tumor suppressors p53 and ARF control ADAR1-driven tumorigenicity in triple negative breast cancer

Abstract Research Overview and Objective Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant challenge. The co-deficiency of tumor suppressors p53 and ARF is a significant genetic signature enriched in TNBC, but it is not yet clear how TNBC is regulated by this genetic alteration. Methods To answer this question, we established p53/ARF-defective murine embryonic fibroblast (MEF) to study the molecular and phenotypic consequences in vitro. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. Results Increased transformation capability was observed in p53/ARF-defective cells, and formation of aggressive mammary tumors was also seen in p53−/−ARF−/− mice. RNA-editing enzyme ADAR1 was identified as a potential mediator for the elevated oncogenic potential. Interestingly, we found that the overexpression of ADAR1 is also prevalent in human TNBC cell lines and patient specimen. Using short hairpin RNA (shRNA) to reduce ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. Different levels of RNA editing of known ADAR1 targets were detected in shRNA-treated human TNBC cell lines, suggesting that ADAR1-mediated RNA editing contributes to TNBC pathogenesis. Implication/Discussion These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, partially through affecting ADAR1-mediated RNA editing. Further understanding of this pathway could shed light on potential vulnerabilities of TNBC and inform the development of personalized therapies based on patients’ genetic signiatures. Citation Format: Che-Pei Kung, Catherine E Kuzmicki, Emily A Bross, Sua Ryu, Eric Freeman, Thwisha Sabloak, Emily R Bramel, Leonard B Maggi Jr, Jason D Weber. Tumor suppressors p53 and ARF control ADAR1-driven tumorigenicity in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-03.

Abstract 4330: Activation of AZIN1 RNA editing facilitates and promotes invasive potential of cancer associated fibroblasts in colorectal cancer

Abstract Colorectal cancer (CRC) is a common malignancy worldwide and remains the second leading cause of cancer-related deaths in Western countries. Emerging evidence demonstrates that such lifestyle choices profoundly impact various epigenetic modifications, and lead to cancer.Adenosine-to-inosine (A-to-I) RNA editing is an epigenetic modification mediated by Adenosine Deaminases that act on the RNA (ADAR), and has recently been discovered to be dysregulated in human cancers. However, the clinical significance and the functional role of RNA editing in cancer-associated fibroblasts (CAFs) remains unclear. By systematically analyzing a large cohort of 627 CRC specimens, we investigated the expression pattern of ADAR1 and the its biological significance on the antizyme inhibitor 1(AZIN1)RNA editing levels, which is one of the most frequently edited genes in cancers. Both ADAR1 expression and AZIN1RNA editing levels were significantly elevated in CRC tissues vs. normal mucosa, and these findings correlated with the increased expression of mesenchymal markers, Vimentin (ρ=0.44) and Fibroblast activation protein (ρ=0.38). ADAR1 was specifically upregulated in consensus molecular subtype 4 (CMS4) CRCs, which have mesenchymal characteristics and associate with poor prognosis.Intriguingly, ADAR1 was overexpressed in both cancer cells and fibroblasts from cancerous lesions. We next assessed whether cancer cells can promote ADAR1 expression and enhance AZIN1RNA editing in fibroblasts. Fibroblasts were cultured in the conditioned medium (CM) derived from CRC cells. Fibroblasts cultured with such CM expressed ADAR1 at significantly higher levels when compared to controls. Likewise, the overexpression of ADAR1 was associated with corresponding increase in AZIN1RNA editing. Overexpression of edited AZIN1RNA enhanced invasiveness of fibroblasts relative to wild-type AZIN1(p=0.0079).Previously the oncogene, ODC, was identified as a downstream target of edited AZIN1. The overexpression of edited AZIN1RNA resulted in upregulation of ODC protein in fibroblasts, confirming that edited AZIN1RNA stabilizes ODC more effectively than its wild-type counterpart in fibroblasts. In the previous study, ODC was reported to be the promoter of invasive potential in fibroblasts. Our results suggest that edited AZIN1RNA may promote invasion of fibroblasts via accumulation of ODC.This study is the first to investigate the clinical significance of ADAR1 expression and the degree of AZIN1RNA editing in colorectal CAFs, along with the determination the functional role of AZIN1RNA editing in this malignancy. Taken together, our study suggests that overexpression of ADAR1 in cancer lesion promotes malignant potential in CRC. We present novel evidence that ADAR1 is overexpressed in fibroblasts and may facilitate invasive potential in cancerous lesions in CRC microenvironment. Citation Format: Sho Takeda, Kunitoshi Shigeyasu, Yoshinaga Okugawa, Kazuhiro Yoshida, Yoshiko Mori, Shuya Yano, Kazuhiro Noma, Yuzo Umeda, Yoshitaka Kondo, Hiroyuki Kishimoto, Fuminori Teraishi, Hiroshi Tazawa, Shunsuke Kagawa, Ajay Goel, Toshiyoshi Fujiwara. Activation of AZIN1 RNA editing facilitates and promotes invasive potential of cancer associated fibroblasts in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4330.

3174 Tumor suppressors p53 and ARF control oncogenic potential of triple-negative breast cancer cells by regulating RNA editing enzyme ADAR1

OBJECTIVES/SPECIFIC AIMS: Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant challenge. The co-deficiency of tumor suppressors p53 and ARF is a significant genetic signature enriched in TNBC, but it is not yet clear how TNBC is regulated by this genetic alteration. METHODS/STUDY POPULATION: To answer this question, we established p53/ARF-defective murine embryonic fibroblast (MEF) to study the molecular and phenotypic consequences in vitro. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. RESULTS/ANTICIPATED RESULTS: Increased transformation capability was observed in p53/ARF-defective cells, and formation of aggressive mammary tumors was also seen in p53-/-ARF-/- mice. RNA-editing enzyme ADAR1 was identified as a potential mediator for the elevated oncogenic potential. Interestingly, we found that the overexpression of ADAR1 is also prevalent in human TNBC cell lines and patient specimen. Using short hairpin RNA (shRNA) to reduce ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. Different levels of RNA editing of known ADAR1 targets were detected in shRNA-treated human TNBC cell lines, suggesting that ADAR1-mediated RNA editing contributes to TNBC pathogenesis. DISCUSSION/SIGNIFICANCE OF IMPACT: These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, partially through affecting ADAR1-mediated RNA editing. Further understanding of this pathway could shed light on potential vulnerabilities of TNBC and inform the development of personalized therapies based on patients’ genetic signiatures.

Aberrant hyperediting of the myeloma transcriptome by ADAR1 confers oncogenicity and is a marker of poor prognosis

AbstractDNA alterations have been extensively reported in multiple myeloma (MM); however, they cannot yet fully explain all the biological and molecular abnormalities in MM, which remains to this day an incurable disease with eventual emergence of refractory disease. Recent years have seen abnormalities at the RNA levels being reported to possess potential biological relevance in cancers. ADAR1-mediated A-to-I editing is an important posttranscriptional mechanism in human physiology, and the biological implication of its abnormality, especially at the global level, is underexplored in MM. In this study, we define the biological implications of A-to-I editing and how it contributes to MM pathogenesis. Here, we identified that the MM transcriptome is aberrantly hyperedited because of the overexpression of ADAR1. These events were associated with patients' survival independent of 1q21 amplifications and could affect patients' responsiveness to different treatment regimes. Our functional assays established ADAR1 to be oncogenic, driving cellular growth and proliferation in an editing-dependent manner. In addition, we identified NEIL1 (base-excision repair gene) as an essential and a ubiquitously edited ADAR1 target in MM. The recoded NEIL1 protein showed defective oxidative damage repair capacity and loss-of-function properties. Collectively, our data demonstrated that ADAR1-mediated A-to-I editing is both clinically and biologically relevant in MM. These data unraveled novel insights into MM molecular pathogenesis at the global RNA level.

2213 Tumor suppressors p53 and ARF control oncogenic potential of triple-negative breast cancer cells by regulating RNA editing enzyme ADAR1

OBJECTIVES/SPECIFIC AIMS: Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant adventure. The deficiency of tumor suppressors p53 and ARF is one of the known genetic signatures enriched in TNBC. Crucial questions remain about how TNBC is regulated by these genetic alterations. METHODS/STUDY POPULATION: In order to address this issue, we established p53/ARF-defective murine embryonic fibroblast and mammary epithelial cell to study the molecular and phenotypic consequences. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. RESULTS/ANTICIPATED RESULTS: Increased proliferation and transformation capability were observed in p53/ARF-defective cells, and an aggressive form of mammary tumor was also seen in p53−/−ARF−/− mice. Gene expression profiling and knock-down experiments using shRNAs were conducted to identify inflammatory marker ISG15 and RNA-editing enzyme ADAR1 as potential culprits for the elevated oncogenic potential. Interestingly, we found that the overexpression of ISG15 and ADAR1 is also prevalent in human TNBC cell lines. Reducing ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. DISCUSSION/SIGNIFICANCE OF IMPACT: These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, likely through regulating ADAR1-mediated RNA modifications. Further understanding of this pathway promises to shed light on genetics-driven vulnerabilities of TNBC and inform development of more effective therapeutic strategies.

The Role of ADAR1 and ADAR2 in the Regulation of miRNA-21 in Idiopathic Pulmonary Fibrosis.

microRNAs (miRNAs) are small non-coding 1RNAs that post-transcriptionally regulate gene expression. Recent evidence shows that adenosine deaminases that act on RNA (ADAR) can edit miRNAs. miRNAs are involved in the development of different diseases, such as idiopathic pulmonary fibrosis (IPF). In IPF, about 40% of the miRNAs are differentially expressed with respect to controls. Among these miRNAs, miRNA-21 has been found over-expressed in IPF and its targets are anti-fibrosing molecules such as PELI1 and SPRY2. The objective of this study is to determine the role of ADAR1 and 2 on the expression of miRNA-21 in human lung fibroblasts trough quantification of gene expression, protein levels, and overexpression of ADAR1 and 2. Six control and six fibrotic primary fibroblast cell cultures were used for RNA extraction, ADAR1, ADAR2, PELI1, SPRY2, miRNA-21, and pri-miRNA-21 expression was measured. Subsequently, two fibrotic fibroblast cultures were used for overexpression of ADAR1 and ADAR2, and they were stimulated with TGFβ1. Real-time PCR and Western blot were performed. ADAR1 is significantly downregulated in IPF fibroblasts; the overexpression of ADAR1 and ADAR2 reestablishes the expression levels of miRNA-21, PELI1, and SPRY2 in fibroblasts of patients with IPF. These changes in the processing of miRNAs have great value in pathology diagnosis, including lung diseases, and play an important role in the understanding of molecular mechanisms involved in the development of different pathologies, as well as representing new therapeutic targets.

Circular RNA expression is suppressed by androgen receptor (AR)-regulated adenosine deaminase that acts on RNA (ADAR1) in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a heterogeneous malignancy as a result of complex genetic and epigenetic alterations. HCC is characterized by a clear gender disparity for which there is lack of a clear mechanistic understanding. Androgen receptor (AR) is thought to be critical for such bias. Meanwhile, the potential function of circular RNA (circRNA), regulated by RNA editing enzyme, remained largely unknown in malignancy till now. By utilizing circRNA microarray survey coupled with in vitro analysis, we analyzed the influence of AR on circRNA expression in HCC. Our results indicated that AR could suppress circRNA expression by upregulating ADAR1 p110. Such effect is because AR served as a transcriptional activator of ADAR1 promoter. More significantly, data collected from our center strongly suggest that ADAR1 expression can effectively predict HCC patients’ prognosis and an abnormal overexpression of ADAR1 is positively correlated with AR in HCC. In addition, we found CircARSP91 (hsa_circ_0085154), one of the circRNAs downregulated by AR in an ADAR1-dependent manner, could inhibit HCC tumor growth both in vitro and in vivo. These findings highlight the fact that AR as a contributing factor for gender disparity in HCC can cause complex consequences though regulation of circRNA expression. Better understanding of the roles of circRNA during HCC initiation and progression will provide a novel angle to develop potential HCC therapies.

Aberrant overexpression of ADAR1 promotes gastric cancer progression by activating mTOR/p70S6K signaling.

ADAR1, one of adenosine deaminases acting on RNA, modulates RNA transcripts through converting adenosine (A) to inosine (I) by deamination. Emerging evidence has implicated that ADAR1 plays an important role in a few of human cancers, however, its expression and physiological significance in gastric cancer remain undefined. In the present study, we demonstrated that ADAR1 was frequently overexpressed in gastric cancer samples by quantitative real-time PCR analysis. In a gastric cancer tissue microarray, ADAR1 staining was closely correlated with tumor stage (P < 0.001) and N classification (P < 0.001). Functional analysis indicated that ADAR1 overexpression promoted cell proliferation and migration in vitro, whereas ADAR1 knockdown resulted in an opposite phenotypes. Furthermore, ADAR1 knockdown also inhibited tumorigenicity and lung metastasis potential of gastric cancer cells in nude mice models. Mechanistically, ADAR1 expression had a significant effect on phosphorylation level of mTOR, p70S kinase, and S6 ribosomal protein, implying its involvement in the regulation of mTOR signaling pathway. We conclude that ADAR1 contributes to gastric cancer development and progression via activating mTOR/p70S6K/S6 ribosomal protein signaling axis. Our findings suggest that ADAR1 may be a valuable biomarker for GC diagnosis and prognosis and may represent a new novel therapeutic opportunities.

Double-stranded-RNA-specific adenosine deaminase 1 (ADAR1) is proposed to contribute to the adaptation of equine infectious anemia virus from horses to donkeys.

Equine infectious anemia virus (EIAV) is a member of the genus Lentivirus of the family Retroviridae. Horses are the most susceptible equids to EIAV infection and are therefore the primary hosts of this virus. In contrast, infected donkeys do not develop clinically active equine infectious anemia (EIA). This phenomenon is similar to what has been observed with HIV-1, which fails to induce AIDS in non-human primates. Interestingly, Shen et al. developed a donkey-tropic pathogenic virus strain (EIAVDV117, DV117) by serially passaging a horse-tropic pathogenic strain, EIAVLN40 (LN40), in donkeys. LN40, which was generated by passaging a field isolate in horses, displayed enhanced virulence in horses but caused no clinical symptoms in donkeys. Infection with DV117 induced acute EIA in nearly 100% of donkeys. Genomic analysis of DV117 revealed a significantly higher frequency of A-to-G substitutions when compared to LN40. Furthermore, detailed analysis of dinucleotide editing showed that A-to-G mutations had a preference for 5'TpA and 5'ApA. These results strongly implicated the activity of the adenosine deaminase, ADAR1, in this type of mutation. Further investigation demonstrated that overexpression of donkey ADAR1 increased A-to-G mutations within the genome of EIAV. Together with our previous finding that multiple mutations in multiple genes are generated in DV117 during its adaptation from horses to donkeys, the present study suggests that ADAR1-induced A-to-G mutations occur during virus adaption to related new hosts contributing to the alteration of EIAV host tropism.

Modifying microRNAs--another piece of the melanoma puzzle.

Modifying microRNAs--another piece of the melanoma puzzle.

Regulation of Self-Renewal through Modulation of Let-7 Stem Cell Regulatory Family of microRNAs in Chronic Myeloid Leukemia Stem Cells

IntroductionChronic myeloid leukemia (CML) was one of the first malignancies shown to be initiated in hematopoietic stem cells by the BCR-ABL1 oncogene and sustained in blast crisis (BC) by progenitor cells that co-opt stem cell properties and behave like leukemia stem cells (BC LSCs). The BCR-ABL fusion oncogene encodes a constitutively active tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitor (TKI) therapy targeting BCR-ABL suppresses CML during the chronic phase (CP), progenitors undergo expansion as a consequence of subsequent genetic and epigenetic alterations that fuel blast crisis transformation, BC LSC generation and TKI resistance. Self-renewing human BC LSCs harbor increased expression of Inflammation responsive adenosine deaminase acting on RNA (ADAR1), which can alter transcript as well as microRNA (miRNA) maturation, splicing and translation by Adenosine (A)-to-Inosine (I) editing of double stranded RNA. miRNAs are a family of small non-coding RNA molecules that regulate gene expression at a post-transcriptional level by inhibiting protein translation and/or reducing mRNA stability. Eukaryotic cells employ miRNAs in diverse biological processes including cell proliferation, differentiation, pluripotency and self-renewal. The stem cell pluripotency RNA binding protein LIN28B plays critical roles in BC transformation of CML. In this study we sought to characterize CML related-oncogenes, such as BCR-ABL, JAK2 and ADAR1, alone or in stromal co-culture in terms of their ability to regulate LSC self-renewal through modulation of let-7 /LIN28B stem cell transcriptional regulatory axis.MethodsMiRNAs were extracted from purified CD34+ cells derived from CP and BC CML patient samples as well as cord blood by RNeasy microKit (QIAGEN) and let-7 expression was evaluated by qRT-PCR using miScript Primer assay (QIAGEN). CD34+ cord blood (n=3) were transduced with lentiviral human BCR-ABL, JAK2, let-7a, wild type ADAR1 and ADAR1 mutant, which lacks a functional deaminase domain. Then, 72 hours after transduction, lentivirally transduced cells were plated on irradiated SL/M2 cells. After 5 days of culture, cells were collected for RNA and microRNA extraction. Transduction efficiency and LIN28B levels were evaluated by qRT-PCR and let-7 expression was quantified by qRT-PCR using miScript primer assay. Hematopoietic Progenitor and Replating assaywere performed on lenti-let-7a-overexpressing CB cells to assess differentiation, survival and self-renewal capacity.ResultsLentiviral overexpression of human BCR-ABL in CD34+ CB did not induce any significant change in let-7 family members and LIN28B expression in absence of stromal co-culture. However, stromal co-culture of BCR-ABL overexpressing CB led to the significant downregulation of members of the let-7 family as well as to upregulation of their target gene LIN28B, thus suggesting that extrinsic microenvironmental cues are necessary for modulating let-7 family levels in presence of BCR-ABL. Notably, qRT-PCR of CB transduced with JAK2 showed significant upregulation of ADAR1 in the absence of stroma, thus suggesting that JAK2 might be a mediator of inflammatory cytokine-driven ADAR1 activation. Lentiviral overexpression of both human JAK2 and ADAR1 significantly reduced the expression of let-7 family members and induced up-regulation of LIN28B. Interestingly, lentiviral overexpression of ADAR1 mutant did not induce any significant change in most let-7 family members. Finally, lentiviral overexpression of let-7a induced significant reduction in survival and self-renewal.ConclusionThese finding suggest that BCR-ABL requires extrinsic signals from the niche to modulate self-renewal of BC LSCs. Conversely, lentiviral JAK2 overexpression induces activation of aberrant RNA editing and subsequent reduction of let-7 family members in the absence of the niche. Interestingly, experiments with ADAR1 mutant, suggest that ADAR1 downregulates most of the let-7 family members in a RNA–editing dependent way manner. In summary these findings suggest a novel mechanism for BC LSC generation that may have utility in prognostication and selective LSCs targeting. DisclosuresJamieson:J&J: Research Funding; Sanofi: Research Funding, Travel Support, Travel Support Other; Roche: Research Funding.

Abstract 1912: ADAR1-mediated microRNA regulation and blast crisis leukemia stem cell generation in chronic myeloid leukemia

Abstract Introduction Chronic myeloid leukemia (CML) was one of the first malignancies shown to be initiated in hematopoietic stem cells by the BCR-ABL1 oncogene and sustained in blast crisis (BC) by progenitor cells that co-opt stem cell properties and behave like leukemia stem cells (BC LSC). The BCR-ABL fusion oncogene encodes a constitutively active tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitor (TKI) therapy targeting BCR-ABL suppresses CML during the chronic phase (CP), progenitors undergo expansion as a consequence of subsequent genetic and epigenetic alterations that fuel blast crisis (BC) transformation, BC LSC generation and TKI resistance. We have recently shown that adenosine to insoine (A-to-I) RNA editing activity, mediated by the adenosine deaminase acting on dsRNAs ADAR1, is enhanced in BC CML and drives leukemic progression by inducing myeloid progenitor reprogramming and enhancing self-renewal. ADAR1 targets double-stranded RNA hairpin-containing loop structures, such as microRNAs (miRNAs). MiRNAs, by operating through base-pairing with complementary sequences within a mRNA molecule lead to mRNA degradation and gene silencing. Emerging evidence suggests miRNAs alterations introduced at the pri-miRNA or pre-miRNA level by A-to-I editing by ADAR1 may be responsible for broad changes in the transcriptome that drives LSC maintenance. However, the overall change in edited miRNA profiles in CP CML compared with BC progenitors is unclear. In this study we aimed to investigate the role of ADAR1 in driving malignant progression by focusing on its regulation of miRNA expression in CML. Methods and Results MicroRNA expression profiles were evaluated in CD34+ cells derived from CP and BC CML patient samples as well as cord blood. MiRNA PCR array (Qiagen) allowed us to identify differentially expressed miRNAs that were subsequently validated by q-PCR with specific primers targeting individual miRNA. We observed the downregulation of 13 miRNA in CML BC compared to CML CP progenitors. Lentiviral overexpression of ADAR1 in CD34+ cord blood cells led to significant downregulation of 17 miRNAs, including members of the let-7 family, and other miRNAs involved in the regulation of self-renewal. Interestingly, BC CD34+ cells showed a miRNA profile similar to that of lentiviral ADAR1 transduced cord blood progenitors. Lentiviral overexpression of ADAR1 in CD34+ chronic phase CML cells, led to a significant downregulation of 14 miRNAs compared with their lentiviral backbone transduced counterparts. Conclusion Our data suggest that ADAR1 contributes to BC LSC generation by downregulating miRNAs that target stem cell regulatory gene products. A further characterization of ADAR-editing related miRNA signatures may enable BC LSC detection and early intervention with combined BCR-ABL1 and ADAR1-targeting strategies that may obviate CML relapse and progression. Citation Format: Maria Zipeto, Qingfei Jiang, Leslie Crews Robertson, Catriona HM Jamieson. ADAR1-mediated microRNA regulation and blast crisis leukemia stem cell generation in chronic myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1912. doi:10.1158/1538-7445.AM2014-1912

Adenosine-to-Inosine RNA Editing Mediated by ADARs in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC), the major histologic form of esophageal cancer, is a heterogeneous tumor displaying a complex variety of genetic and epigenetic changes. Aberrant RNA editing of adenosine-to-inosine (A-to-I), as it is catalyzed by adenosine deaminases acting on RNA (ADAR), represents a common posttranscriptional modification in certain human diseases. In this study, we investigated the status and role of ADARs and altered A-to-I RNA editing in ESCC tumorigenesis. Among the three ADAR enzymes expressed in human cells, only ADAR1 was overexpressed in primary ESCC tumors. ADAR1 overexpression was due to gene amplification. Patients with ESCC with tumoral overexpression of ADAR1 displayed a poor prognosis. In vitro and in vivo functional assays established that ADAR1 functions as an oncogene during ESCC progression. Differential expression of ADAR1 resulted in altered gene-specific editing activities, as reflected by hyperediting of FLNB and AZIN1 messages in primary ESCC. Notably, the edited form of AZIN1 conferred a gain-of-function phenotype associated with aggressive tumor behavior. Our findings reveal that altered gene-specific A-to-I editing events mediated by ADAR1 drive the development of ESCC, with potential implications in diagnosis, prognosis, and treatment of this disease.