The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Contribution of A-to-I RNA editing, M6A RNA Methylation, and Alternative Splicing to physiological brain aging and neurodegenerative diseases.

Aging is a physiological and progressive phenomenon in all organisms' life cycle, characterized by the accumulation of degenerative processes triggered by several alterations within molecular pathways. These changes compromise cell fate, resulting in the loss of functions in tissues throughout the body, including the brain. Physiological brain aging has been linked to structural and functional alterations, as well as to an increased risk of neurodegenerative diseases. Post-transcriptional RNA modifications modulate mRNA coding properties, stability, translatability, expanding the coding capacity of the genome, and are involved in all cellular processes. Among mRNA post-transcriptional modifications, the A-to-I RNA editing, m6A RNA Methylation and Alternative Splicing play a critical role in all the phases of a neuronal cell life cycle and alterations in their mechanisms of action significantly contribute to aging and neurodegeneration. Here we review our current understanding of the contribution of A-to-I RNA editing, m6A RNA Methylation, and Alternative Splicing to physiological brain aging process and neurodegenerative diseases.

Impact of age and gender on glioblastoma onset, progression, and management.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, while its frequency in pediatric patients is 10-15%. For this reason, age is considered one of the major risk factors for the development of GBM, as it correlates with cellular aging phenomena involving glial cells and favoring the process of tumor transformation. Gender differences have been also identified, as the incidence of GBM is higher in males than in females, coupled with a worse outcome. In this review, we analyze age- and gender- dependent differences in GBM onset, mutational landscape, clinical manifestations, and survival, according to the literature of the last 20 years, focusing on the major risk factors involved in tumor development and on the mutations and gene alterations most frequently found in adult vs young patients and in males vs females. We then highlight the impact of age and gender on clinical manifestations and tumor localization and their involvement in the time of diagnosis and in determining the tumor prognostic value.

Sex differences in antioxidant defence and the regulation of redox homeostasis in physiology and pathology.

Reactive oxygen species (ROS) is a term that defines a group of unstable compounds derived from exogenous sources or endogenous metabolism. Under physiological conditions, low levels of ROS play a key role in the regulation of signal transduction- or transcription-mediated cellular responses. In contrast, excessive and uncontrolled loading of ROS results in a pathological state known as oxidative stress (OS), a leading contributor to aging and a pivotal factor for the onset and progression of many disorders. Evolution has endowed cells with an antioxidant system involved in stabilizing ROS levels to a specific threshold, maintaining ROS-induced signalling function and limiting negative side effects. In mammals, a great deal of evidence indicates that females defence against ROS is more proficient than males, determining a longer lifespan and lower incidence of most chronic diseases. In this review, we will summarize the most recent sex-related differences in the regulation of redox homeostasis. We will highlight the peculiar aspects of the antioxidant defence in sex-biased diseases whose onset or progression is driven by OS, and we will discuss the molecular, genetic, and evolutionary determinants of female proficiency to cope with ROS.

Direct CD32 T-cell cytotoxicity: implications for breast cancer prognosis and treatment.

The FcγRII (CD32) ligands are IgFc fragments and pentraxins. The existence of additional ligands is unknown. We engineered T cells with human chimeric receptors resulting from the fusion between CD32 extracellular portion and transmembrane CD8α linked to CD28/ζ chain intracellular moiety (CD32-CR). Transduced T cells recognized three breast cancer (BC) and one colon cancer cell line among 15 tested in the absence of targeting antibodies. Sensitive BC cell conjugation with CD32-CR T cells induced CD32 polarization and down-regulation, CD107a release, mutual elimination, and proinflammatory cytokine production unaffected by human IgGs but enhanced by cetuximab. CD32-CR T cells protected immunodeficient mice from subcutaneous growth of MDA-MB-468 BC cells. RNAseq analysis identified a 42 gene fingerprint predicting BC cell sensitivity and favorable outcomes in advanced BC. ICAM1 was a major regulator of CD32-CR T cell-mediated cytotoxicity. CD32-CR T cells may help identify cell surface CD32 ligand(s) and novel prognostically relevant transcriptomic signatures and develop innovative BC treatments.

ADAR2 Protein Is Associated with Overall Survival in GBM Patients and Its Decrease Triggers the Anchorage-Independent Cell Growth Signature.

BACKGROUND: Epitranscriptomic mechanisms, such as A-to-I RNA editing mediated by ADAR deaminases, contribute to cancer heterogeneity and patients' stratification. ADAR enzymes can change the sequence, structure, and expression of several RNAs, affecting cancer cell behavior. In glioblastoma, an overall decrease in ADAR2 RNA level/activity has been reported. However, no data on ADAR2 protein levels in GBM patient tissues are available; and most data are based on ADARs overexpression experiments. METHODS: We performed IHC analysis on GBM tissues and correlated ADAR2 levels and patients' overall survival. We silenced ADAR2 in GBM cells, studied cell behavior, and performed a gene expression/editing analysis. RESULTS: GBM tissues do not all show a low/no ADAR2 level, as expected by previous studies. Although, different amounts of ADAR2 protein were observed in different patients, with a low level correlating with a poor patient outcome. Indeed, reducing the endogenous ADAR2 protein in GBM cells promotes cell proliferation and migration and changes the cell's program to an anchorage-independent growth mode. In addition, deep-seq data and bioinformatics analysis indicated multiple RNAs are differently expressed/edited upon siADAR2. CONCLUSION: ADAR2 protein is an important deaminase in GBM and its amount correlates with patient prognosis.

miRNome and Proteome Profiling of Small Extracellular Vesicles Secreted by Human Glioblastoma Cell Lines and Primary Cancer Stem Cells.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. Despite available therapeutic interventions, it is very difficult to treat, and a cure is not yet available. The intra-tumoral GBM heterogeneity is a crucial factor contributing to poor clinical outcomes. GBM derives from a small heterogeneous population of cancer stem cells (CSCs). In cancer tissue, CSCs are concentrated within the so-called niches, where they progress from a slowly proliferating phase. CSCs, as most tumor cells, release extracellular vesicles (EVs) into the surrounding microenvironment. To explore the role of EVs in CSCs and GBM tumor cells, we investigated the miRNA and protein content of the small EVs (sEVs) secreted by two GBM-established cell lines and by GBM primary CSCs using omics analysis. Our data indicate that GBM-sEVs are selectively enriched for miRNAs that are known to display tumor suppressor activity, while their protein cargo is enriched for oncoproteins and tumor-associated proteins. Conversely, among the most up-regulated miRNAs in CSC-sEVs, we also found pro-tumor miRNAs and proteins related to stemness, cell proliferation, and apoptosis. Collectively, our findings support the hypothesis that sEVs selectively incorporate different miRNAs and proteins belonging both to fundamental processes (e.g., cell proliferation, cell death, stemness) as well as to more specialized ones (e.g., EMT, membrane docking, cell junction organization, ncRNA processing).

MAPK activation drives male and female mouse teratocarcinomas from late primordial germ cells.

Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior.

ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair.

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

ADAR1 is a new target of METTL3 and plays a pro-oncogenic role in glioblastoma by an editing-independent mechanism.

BACKGROUND: N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) RNA editing are two of the most abundant RNA modification events affecting adenosines in mammals. Both these RNA modifications determine mRNA fate and play a pivotal role in tumor development and progression. RESULTS: Here, we show that METTL3, upregulated in glioblastoma, methylates ADAR1 mRNA and increases its protein level leading to a pro-tumorigenic mechanism connecting METTL3, YTHDF1, and ADAR1. We show that ADAR1 plays a cancer-promoting role independently of its deaminase activity by binding CDK2 mRNA, underlining the importance of ADARs as essential RNA-binding proteins for cell homeostasis as well as cancer progression. Additionally, we show that ADAR1 knockdown is sufficient to strongly inhibit glioblastoma growth in vivo. CONCLUSIONS: Hence, our findings underscore METTL3/ADAR1 axis as a novel crucial pathway in cancer progression that connects m6A and A-to-I editing post-transcriptional events.

MicroRNA Editing Detection and Function: A Combined In Silico and Experimental Approach for the Identification and Validation of Putative Oncogenic Targets.

MicroRNAs (miRNAs) are a class of ~22 nt noncoding RNAs playing essential roles in the post-transcriptional regulation of gene expression, cell proliferation, and cell differentiation and are often found deregulated in several diseases including cancer.The A-to-I RNA editing, mediated by ADAR enzymes, is a diffuse post-transcriptional mechanism that converts the genetically coded adenosine (A) into inosine (I) at the RNA level. Among different RNA targets, the ADAR enzymes can also edit miRNA precursors. Specifically, a single nucleotide change (A/I) lying within the mature miRNA can alter the miRNA binding specificity and redirect the edited miRNA to a different mRNA target. In several cancer types a consistent deregulation of A-to-I RNA editing machinery also involves important miRNAs (either oncomiRs or tumor-suppressor miRNAs). Herein we describe a combined in silico and experimental approach for the detection of edited miRNAs and the identification and validation of their target genes potentially involved in cancer progression or invasion.

Aptamer-Based In Vivo Therapeutic Targeting of Glioblastoma.

Glioblastoma (GBM) is the most aggressive, infiltrative, and lethal brain tumor in humans. Despite the extensive advancement in the knowledge about tumor progression and treatment over the last few years, the prognosis of GBM is still very poor due to the difficulty of targeting drugs or anticancer molecules to GBM cells. The major challenge in improving GBM treatment implicates the development of a targeted drug delivery system, capable of crossing the blood-brain barrier (BBB) and specifically targeting GBM cells. Aptamers possess many characteristics that make them ideal novel therapeutic agents for the treatment of GBM. They are short single-stranded nucleic acids (RNA or ssDNA) able to bind to a molecular target with high affinity and specificity. Several GBM-targeting aptamers have been developed for imaging, tumor cell isolation from biopsies, and drug/anticancer molecule delivery to the tumor cells. Due to their properties (low immunogenicity, long stability, and toxicity), a large number of aptamers have been selected against GBM biomarkers and tested in GBM cell lines, while only a few of them have also been tested in in vivo models of GBM. Herein, we specifically focus on aptamers tested in GBM in vivo models that can be considered as new diagnostic and/or therapeutic tools for GBM patients' treatment.

Type 5 phosphodiesterase (PDE5) and the vascular tree: From embryogenesis to aging and disease.

Vascular tree development depends on the timely differentiation of endothelial and vascular smooth muscle cells. These latter are key players in the formation of the vascular scaffold that offers resistance to the blood flow. This review aims at providing an overview on the role of PDE5, the cGMP-specific phosphodiesterase that historically attracted much attention for its involvement in male impotence, in the regulation of vascular smooth muscle cell function. The overall goal is to underscore the importance of PDE5 expression and activity in this cell type in the context of the organs where its function has been extensively studied.

Deregulation of TLR4 signaling pathway characterizes Bicuspid Aortic valve syndrome.

Bicuspid aortic valve (BAV) disease is recognized to be a syndrome with a complex and multifaceted pathophysiology. Its progression is modulated by diverse evolutionary conserved pathways, such as Notch-1 pathway. Emerging evidence is also highlighting the key role of TLR4 signaling pathway in the aortic valve pathologies and their related complications, such as sporadic ascending aorta aneurysms (AAA). Consistent with these observations, we aimed to evaluate the role of TLR4 pathway in both BAV disease and its common complication, such as AAA. To this aim, 70 subjects with BAV (M/F 50/20; mean age: 58.8 ± 14.8 years) and 70 subjects with tricuspid aortic valve (TAV) (M/F 35/35; mean age: 69.1 ± 12.8 years), with and without AAA were enrolled. Plasma assessment, tissue and gene expression evaluations were performed. Consistent with data obtained in the previous study on immune clonotypic T and B altered responses, we found reduced levels of systemic TNF-α, IL-1, IL-6, IL-17 cytokines in BAV cases, either in the presence or absence of AAA, than TAV cases (p < 0.0001 by ANOVA test). Interestingly, we also detected reduced levels of s-TLR4 in BAV cases with or without AAA in comparison to the two groups of TAV subjects (p < 0.0001 by ANOVA test). These results may suggest a deregulation in the activity or in the expression of TLR4 signaling pathway in all BAV cases. Portrait of these data is, indeed, the significantly decreased gene expression of inflammatory cytokines and TLR4, in both normal and aneurysmatic tissue samples, from BAV with AAA than TAV with AAA. In conclusion, our study demonstrates that subjects with BAV display a significant deregulation of TLR4 signaling pathway paralleled by a deregulation of Notch-1 pathway, as previously showed. This data suggests that the crosstalk between the Notch-1 and TLR4 signaling pathways may play a crucial role in both physiological embryological development, and homeostasis and functionality of aortic valve in adult life.

Regulation of PDE5 expression in human aorta and thoracic aortic aneurysms.

Aneurysms and dissections affecting thoracic aorta are associated with smooth muscle cell (SMC) dysfunction. NO/cGMP signaling pathway in smooth muscle cells has been shown to be affected in sporadic thoracic aortic aneurysms. We analyzed the mRNA levels of PDE5, a cGMP-hydrolyzing enzyme highly expressed in aortic SMCs, that regulates arterious vascular tone by lowering cGMP levels. We found that aortic tissue obtained from Marfan, tricuspid and bicuspid thoracic aneurysms expressed lower levels of PDE5 mRNA compared to control aortas. In particular, we found that affected aortas showed lower levels of all the PDE5A isoforms, compared to control aortas. Transfection of vascular SMCs (VSMCs) with NOTCH3 activated domain (NICD3) induced the expression of PDE5A1 and A3 protein isoforms, but not that of the corresponding mRNAs. VSMC stimulation with GSNO, a nitric oxide analogue or with 8-br-cGMP, but not with 8-br-cAMP, up-regulated PDE5 and NOTCH-3 protein levels, indicating a negative feedback loop to protect the arterial wall from excessive relaxation. Finally, we found that PDE5 is expressed early during human aorta development, suggesting that if loss of function mutations of PDE5 occur, they might potentially affect aortic wall development.

AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.

Murine thymoma viral oncogene homolog (AKT) kinases target both cytosolic and nuclear substrates for phosphorylation. Whereas the cytosolic substrates are known to be closely associated with the regulation of apoptosis and autophagy or metabolism and protein synthesis, the nuclear substrates are, for the most part, poorly understood. To better define the role of nuclear AKT, potential AKT substrates were isolated from the nuclear lysates of leukemic cell lines using a phosphorylated AKT substrate antibody and identified in tandem mass spectrometry. Among the proteins identified was adenosine deaminase acting on RNA (ADAR)1p110, the predominant nuclear isoform of the adenosine deaminase acting on double-stranded RNA. Coimmunoprecipitation studies and in vitro kinase assays revealed that AKT-1, -2, and -3 interact with both ADAR1p110 and ADAR2 and phosphorylate these RNA editases. Using site-directed mutagenesis of suspected AKT phosphorylation sites, AKT was found to primarily phosphorylate ADAR1p110 and ADAR2 on T738 and T553, respectively, and overexpression of the phosphomimic mutants ADAR1p110 (T738D) and ADAR2 (T553D) resulted in a 50-100% reduction in editase activity. Thus, activation of AKT has a direct and major impact on RNA editing.-Bavelloni, A., Focaccia, E., Piazzi, M., Raffini, M., Cesarini, V., Tomaselli, S., Orsini, A., Ratti, S., Faenza, I., Cocco, L., Gallo, A., Blalock, W. L. AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.

Dynamic inosinome profiles reveal novel patient stratification and gender-specific differences in glioblastoma.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is an essential post-transcriptional mechanism mediated by ADAR enzymes that have been recently associated with cancer. RESULTS: Here, we characterize the inosinome signature in normal brain and de novo glioblastoma (GBM) using new metrics that re-stratify GBM patients according to their editing profiles and indicate this post-transcriptional event as a possible molecular mechanism for sexual dimorphism in GBM. We find that over 85% of de novo GBMs carry a deletion involving the genomic locus of ADAR3, which is specifically expressed in the brain. By analyzing RNA editing and patient outcomes, an intriguing gender-dependent link appears, with high editing of Alus shown to be beneficial only in male patients. We propose an inosinome-based molecular stratification of GBM patients that identifies two different GBM subgroups, INO-1 and INO-2, which can identify novel high-risk gender-specific patient groups for which more aggressive treatments may be necessary. CONCLUSIONS: Our data provide a detailed picture of RNA editing landscape in normal brain and GBM, exploring A-to-I RNA editing regulation, disclosing unexpected editing implications for GBM patient stratification and identification of gender-dependent high-risk patients, and suggesting COG3 I/V as an eligible site for future personalized targeted gene therapy.

The adaptive potential of RNA editing-mediated miRNA-retargeting in cancer.

A-to-I RNA editing is a post-transcriptional mechanism that converts the genomically coded Adenosine (A) into Inosine (I) at the RNA level. This type of RNA editing is the most frequent in humans and is mediated by the ADAR enzymes. RNA editing can alter the genetic code of mRNAs, but also affect the functions of noncoding RNAs such as miRNAs. Recent studies have identified thousands of microRNA editing events in different cancer types. However, the important role played by miRNA-editing in cancer has been reported for just a few microRNAs. Herein, we recapitulate the current studies on cancer-related microRNA editing and discuss their importance in tumor growth and progression. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

ADAR2/miR-589-3p axis controls glioblastoma cell migration/invasion.

Recent studies have reported the emerging role of microRNAs (miRNAs) in human cancers. We systematically characterized miRNA expression and editing in the human brain, which displays the highest number of A-to-I RNA editing sites among human tissues, and in de novo glioblastoma brain cancer. We identified 299 miRNAs altered in their expression and 24 miRNAs differently edited in human brain compared to glioblastoma tissues. We focused on the editing site within the miR-589-3p seed. MiR-589-3p is a unique miRNA almost fully edited (∼100%) in normal brain and with a consistent editing decrease in glioblastoma. The edited version of miR-589-3p inhibits glioblastoma cell proliferation, migration and invasion, while the unedited version boosts cell proliferation and motility/invasion, thus being a potential cancer-promoting factor. We demonstrated that the editing of this miRNA is mediated by ADAR2, and retargets miR-589-3p from the tumor-suppressor PCDH9 to ADAM12, which codes for the metalloproteinase 12 promoting glioblastoma invasion. Overall, our study dissects the role of a unique brain-specific editing site within miR-589-3p, with important anticancer features, and highlights the importance of RNA editing as an essential player not only for diversifying the genomic message but also for correcting not-tolerable/critical genomic coding sites.