Angela Gallo

ADAR2-editing activity inhibits glioblastoma growth through the modulation of the CDC14B/Skp2/p21/p27 axis.

Grade IV astrocytoma or glioblastoma multiforme (GBM) is one of the most aggressive and lethal tumors affecting humans. ADAR2-mediated A-to-I RNA editing, an essential post-transcriptional modification event in brain, is impaired in GBMs and astrocytoma cell lines. However, the role of ADAR2 editing in astrocytomas remains to be defined. Here, we show that ADAR2 editing rescue in astrocytomas prevents tumor growth in vivo and modulates an important cell cycle pathway involving the Skp2/p21/p27 proteins, often altered in glioblastoma. We demonstrate that ADAR2 deaminase activity is essential to inhibit tumor growth. Indeed, we identify the phosphatase CDC14B, which acts upstream of the Skp2/p21/p27 pathway, as a novel and critical ADAR2 target gene involved in glioblastoma growth. Specifically, ADAR2-mediated editing on CDC14B pre-mRNA increases its expression with a consequent reduction of the Skp2 target protein, as shown both in vitro and in vivo. We found that, compared to normal brain, both CDC14B editing and expression are progressively impaired in astrocytomas from grade I to IV, being very low in GBMs. These findings (1) demonstrate that post-transcriptional A-to-I RNA editing might be crucial for glioblastoma pathogenesis, (2) identify ADAR2-editing enzyme as a novel candidate tumor suppressor gene and (3) provide proof of principle that ADAR2 or its substrates may represent a suitable target(s) for possible novel, more effective and less toxic approaches to the treatment of GBMs.

A-to-I RNA editing and cancer From pathology to basic science

In eukaryotes mRNA transcripts are extensively processed by different post-transcriptional events such as alternative splicing and RNA editing in order to generate many different mRNAs from the same gene, increasing the transcriptome and then the proteome. The most frequent RNA editing mechanism in mammals involves the conversion of specific adenosines into inosines by the ADAR family of enzymes. This editing event can change both the sequence and the secondary structures of RNA molecules, with important consequences on both the final proteins and regulatory RNAs. Alteration in RNA editing has been connected to numerous human pathologies and recent studies have demonstrated its importance in tumor progression.

Modulation of microRNA editing, expression and processing by ADAR2 deaminase in glioblastoma

BackgroundADAR enzymes convert adenosines to inosines within double-stranded RNAs, including microRNA (miRNA) precursors, with important consequences on miRNA retargeting and expression. ADAR2 activity is impaired in glioblastoma and its rescue has anti-tumoral effects. However, how ADAR2 activity may impact the miRNome and the progression of glioblastoma is not known.ResultsBy integrating deep-sequencing and array approaches with bioinformatics analyses and molecular studies, we show that ADAR2 is essential to edit a small number of mature miRNAs and to significantly modulate the expression of about 90 miRNAs in glioblastoma cells. Specifically, the rescue of ADAR2 activity in cancer cells recovers the edited miRNA population lost in glioblastoma cell lines and tissues, and rebalances expression of onco-miRNAs and tumor suppressor miRNAs to the levels observed in normal human brain. We report that the major effect of ADAR2 is to reduce the expression of a large number of miRNAs, most of which act as onco-miRNAs. ADAR2 can edit miR-222/221 and miR-21 precursors and decrease the expression of the corresponding mature onco-miRNAs in vivo and in vitro, with important effects on cell proliferation and migration.ConclusionsOur findings disclose an additional layer of complexity in miRNome regulation and provide information to better understand the impact of ADAR2 editing enzyme in glioblastoma. We propose that ADAR2 is a key factor for maintaining edited-miRNA population and balancing the expression of several essential miRNAs involved in cancer.

ADAR2 editing enzyme is a novel human immunodeficiency virus-1 proviral factor

The adenosine deaminases acting on RNA (ADAR) enzymes catalyse conversion of adenosine to inosine in dsRNA. A positive effect of ADAR1 on human immunodeficiency virus type 1 (HIV-1) replication has recently been reported. Here, we show that another ADAR enzyme, ADAR2, positively affects the replication process of HIV-1. We found that, analogously to ADAR1, ADAR2 enhances the release of progeny virions by an editing-dependent mechanism. However, differently from the ADAR1 enzyme, ADAR2 does not increase the infectious potential of the virus. Importantly, downregulation of ADAR2 in Jurkat cells significantly impairs viral replication. Therefore, ADAR2 shares some but not all proviral functions of ADAR1. These results suggest a novel role of ADAR2 as a viral regulator.

The RNA editing enzymes ADARs: mechanism of action and human disease

A-to-I RNA editing is a ubiquitous and crucial molecular mechanism able to convert adenosines into inosines (then read as guanosines by several intracellular proteins/enzymes) within RNA molecules, changing the genomic information. The A-to-I deaminase enzymes (ADARs), which modify the adenosine, can alter the splicing and translation machineries, the double-stranded RNA structures and the binding affinity between RNA and RNA-binding proteins. ADAR activity is an essential mechanism in mammals and altered editing has been associated with several human diseases. Many efforts are now being concentrated on modifying ADAR activity in vivo in an attempt to correct RNA editing dysfunction. Concomitantly, ongoing studies aim to show the way that the ADAR deaminase domain can be used as a possible new tool, an intracellular Trojan horse, for the correction of heritage diseases not related to RNA editing events.

Oligophrenin-1 (OPHN1), a gene involved in X-linked intellectual disability, undergoes RNA editing and alternative splicing during human brain development.

Oligophrenin-1 (OPHN1) encodes for a Rho-GTPase-activating protein, important for dendritic morphogenesis and synaptic function. Mutations in this gene have been identified in patients with X-linked intellectual disability associated with cerebellar hypoplasia. ADAR enzymes are responsible for A-to-I RNA editing, an essential post-transcriptional RNA modification contributing to transcriptome and proteome diversification. Specifically, ADAR2 activity is essential for brain development and function. Herein, we show that the OPHN1 transcript undergoes post-transcriptional modifications such as A-to-I RNA editing and alternative splicing in human brain and other tissues. We found that OPHN1 editing is detectable already at the 18th week of gestation in human brain with a boost of editing at weeks 20 to 33, concomitantly with OPHN1 expression increase and the appearance of a novel OPHN1 splicing isoform. Our results demonstrate that multiple post-transcriptional events occur on OPHN1, a gene playing an important role in brain function and development.

ADAR2 editing activity in newly diagnosed versus relapsed pediatric high-grade astrocytomas

BackgroundHigh-grade (WHO grade III and IV) astrocytomas are aggressive malignant brain tumors affecting humans with a high risk of recurrence in both children and adults. To date, limited information is available on the genetic and molecular alterations important in the onset and progression of pediatric high-grade astrocytomas and, even less, on the prognostic factors that influence long-term outcome in children with recurrence. A-to-I RNA editing is an essential post-transcriptional mechanism that can alter the nucleotide sequence of several RNAs and is mediated by the ADAR enzymes. ADAR2 editing activity is particularly important in mammalian brain and is impaired in both adult and pediatric high-grade astrocytomas. Moreover, we have recently shown that the recovered ADAR2 activity in high-grade astrocytomas inhibits in vivo tumor growth. The aim of the present study is to investigate whether changes may occur in ADAR2-mediated RNA editing profiles of relapsed high-grade astrocytomas compared to their respective specimens collected at diagnosis, in four pediatric patients.MethodsTotal RNAs extracted from all tumor samples and controls were tested for RNA editing levels (by direct sequencing on cDNA pools) and for ADAR2 mRNA expression (by qRT-PCR).ResultsA significant loss of ADAR2-editing activity was observed in the newly diagnosed and recurrent astrocytomas in comparison to normal brain. Surprisingly, we found a substantial rescue of ADAR2 editing activity in the relapsed tumor of the only patient showing prolonged survival.ConclusionsHigh-grade astrocytomas display a generalized loss of ADAR2-mediated RNA editing at both diagnosis and relapse. However, a peculiar Case, in complete remission of disease, displayed a total rescue of RNA editing at relapse, intriguingly suggesting ADAR2 activity/expression as a possible marker for long-term survival of patients with high-grade astrocytomas.

Massive transcriptome sequencing of human spinal cord tissues provides new insights into motor neuron degeneration in ALS

ALS is a devastating and debilitating human disease characterized by the progressive death of upper and lower motor neurons. Although much effort has been made to elucidate molecular determinants underlying the onset and progression of the disorder, the causes of ALS remain largely unknown. In the present work, we have deeply sequenced whole transcriptome from spinal cord ventral horns of post-mortem ALS human donors affected by the sporadic form of the disease (which comprises ~90% of the cases but which is less investigated than the inherited form of the disease). We observe 1160 deregulated genes including 18 miRNAs and show that down regulated genes are mainly of neuronal derivation while up regulated genes have glial origin and tend to be involved in neuroinflammation or cell death. Remarkably, we find strong deregulation of SNAP25 and STX1B at both mRNA and protein levels suggesting impaired synaptic function through SNAP25 reduction as a possible cause of calcium elevation and glutamate excitotoxicity. We also note aberrant alternative splicing but not disrupted RNA editing.

The role of HCMV and HIV-1 MicroRNAs: Processing, and mechanisms of action during viral infection

Viruses infect host cells releasing their genome (DNA or RNA) containing all information needed to replicate themselves. The viral genome takes control of the cells and helps the virus to evade the host immune system. Some viruses alter the functions of infected cells without killing them. In some cases infected cells lose control over normal cell proliferation and becomes cancerous. Viruses, such as HCMV and HIV-1, may leave their viral genome in the host cells for a certain period (latency) and begin to replicate when the cells are stressed causing diseases. HCMV and HIV-1 have developed multiple strategies to avoid recognition and elimination by the host’s immune system. These strategies rely on viral products that mimic specific components of the host cells to prevent immune recognition of virally infected cells. In addition to viral proteins, viruses encode short non-coding RNAs (vmiRNAs) that regulate both viral and host cellular transcripts to favor viral infection and actively curtail the host’s antiviral immune response. In this review, we will give an overview of the general functions of microRNAs generated by HCMV and HIV-1, their processing and interaction with the host’s immune system.

Peripheral medulloepithelioma: a rare tumor with a potential target therapy

BackgroundMedulloepithelioma (ME) is a rare embryonal tumor predominantly located in the eye or in the central nervous system without an established treatment.Case presentationWe report of a case of a localized peripheral ME treated with conventional and high dose chemotherapy, surgery and local radiotherapy. At relapse, the tumor tissue revealed a different molecular signature compared to the initial tumor mass. This molecular signature revealed a high expression of platelet derived growth factor receptor (PDGFR). Sorafenib plus irinotecan and temozolomide was started with a 5xa0month progression free survival.ConclusionOur experience suggests a possible role of sorafenib or different PDGFR inhibitors in ME. Targeting treatment could represent an adjuvant and/or alternative therapy for ME and other rare tumors.