ADAR3 modulates neuronal differentiation and regulates mRNA stability and translation.

ADAR3 is a catalytically inactive member of the family of adenosine deaminases acting on RNA (ADARs). Here we have investigated its function in the context of the developing mouse brain. The expression of ADAR3 gradually increases throughout embryogenesis and drops after birth. Using primary cortical neurons, we show that ADAR3 is only expressed in a subpopulation of in vitro differentiated neurons, which suggests specific functions rather than being a general regulator of ADAR editing in the brain. The analysis of the ADAR3 interactome suggested a role in mRNA stability and translation, and we show that expression of ADAR3 in a neuronal cell line that is otherwise ADAR3-negative changes the expression and stability of a large number of mRNAs. Notably, we show that ADAR3 associates with polysomes and inhibits translation. We propose that ADAR3 binds to target mRNAs and stabilizes them in non-productive polysome complexes. Interestingly, the expression of ADAR3 downregulates genes related to neuronal differentiation and inhibits neurofilament outgrowth in vitro. In summary, we propose that ADAR3 negatively regulates neuronal differentiation, and that it does so by regulating mRNA stability and translation in an editing-independent manner.

ADAR-Mediated A>I(G) RNA Editing in the Genotoxic Drug Response of Breast Cancer.

Epitranscriptomics is a field that delves into post-transcriptional changes. Among these modifications, the conversion of adenosine to inosine, traduced as guanosine (A>I(G)), is one of the known RNA-editing mechanisms, catalyzed by ADARs. This type of RNA editing is the most common type of editing in mammals and contributes to biological diversity. Disruption in the A>I(G) RNA-editing balance has been linked to diseases, including several types of cancer. Drug resistance in patients with cancer represents a significant public health concern, contributing to increased mortality rates resulting from therapy non-responsiveness and disease progression, representing the greatest challenge for researchers in this field. The A>I(G) RNA editing is involved in several mechanisms over the immunotherapy and genotoxic drug response and drug resistance. This review investigates the relationship between ADAR1 and specific A>I(G) RNA-edited sites, focusing particularly on breast cancer, and the impact of these sites on DNA damage repair and the immune response over anti-cancer therapy. We address the underlying mechanisms, bioinformatics, and in vitro strategies for the identification and validation of A>I(G) RNA-edited sites. We gathered databases related to A>I(G) RNA editing and cancer and discussed the potential clinical and research implications of understanding A>I(G) RNA-editing patterns. Understanding the intricate role of ADAR1-mediated A>I(G) RNA editing in breast cancer holds significant promise for the development of personalized treatment approaches tailored to individual patients' A>I(G) RNA-editing profiles.

ADAR1- and ADAR2-mediated regulation of maturation and targeting of miR-376b to modulate GABA neurotransmitter catabolism.

miRNAs are short noncoding RNA molecules that regulate gene expression by inhibiting translation or inducing degradation of target mRNAs. miRNAs are often expressed as polycistronic transcripts, so-called miRNA clusters, containing several miRNA precursors. The largest mammalian miRNA cluster, the miR-379-410 cluster, is expressed primarily during embryonic development and in the adult brain; however, downstream regulation of this cluster is not well understood. Here, we investigated adenosine deamination to inosine (RNA editing) in the miR-379-410 cluster by adenosine deaminase acting on RNA (ADAR) enzymes as a possible mechanism modulating the expression and activity of these miRNAs in a brain-specific manner. We show that the levels of editing in the majority of mature miRNAs are lower than the editing levels of the corresponding site in primary miRNA precursors. However, for one miRNA, miR-376b-3p, editing was significantly higher in the mature form than in the primary precursor. We found miR-376b-3p maturation is negatively regulated by ADAR2 in an editing activity-independent manner, whereas ADAR1-mediated and ADAR2-mediated editing were observed to be competitive. In addition, the edited miR-376b-3p targets a different set of mRNAs than unedited miR-376b-3p, including 4-aminobutyrate aminotransferase, encoding the enzyme responsible for the catabolism of the neurotransmitter gamma aminobutyric acid (GABA). Expression of edited miR-376b-3p led to increased intracellular GABA levels as well as increased cell surface presentation of GABA type A receptors. Our results indicate that both editing and editing-independent effects modulate the expression of miR-376b-3p, with the potential to regulate GABAergic signaling in the brain.

TRPM4 channel is involved in regulating epithelial to mesenchymal transition, migration, and invasion of prostate cancer cell lines.

Transient Receptor Potential Melastatin 4 (TRPM4) is a Ca2+ -activated and voltage-dependent monovalent cation channel, which depolarizes the plasma cell membrane, thereby modulating Ca2+ influx across Ca2+ -permeable pathways. TRPM4 is involved in different physiological processes such as T cell activation and the migration of endothelial and certain immune cells. Overexpression of this channel has been reported in various types of tumors including prostate cancer. In this study, a significant overexpression of TRPM4 was found only in samples from cancer with a Gleason score higher than 7, which are more likely to spread. To evaluate whether TRPM4 overexpression was related to the spreading capability of tumors, TRPM4 was knockdown by using shRNAs in PC3 prostate cancer cells and the effect on cellular migration and invasion was analyzed. PC3 cells with reduced levels of TRPM4 (shTRPM4) display a decrease of the migration/invasion capability. A reduction in the expression of Snail1, a canonical epithelial to mesenchymal transition (EMT) transcription factor, was also observed. Consistently, these cells showed a significant change in the expression of key EMT markers such as MMP9, E-cadherin/N-cadherin, and vimentin, indicating a partial reversion of the EMT process. Whereas, the overexpression of TRPM4 in LnCaP cells resulted in increased levels of Snail1, reduction in the expression of E-cadherin and increase in their migration potential. This study suggests a new and indirect mechanism of regulation of migration/invasion process by TRPM4 in prostate cancer cells, by inducing the expression of Snail1 gene and consequently, increasing the EMT.

Mucin 5B, carbonic anhydrase 9 and claudin 18 are potential theranostic markers of gallbladder carcinoma.

AIMS: Gallbladder cancer (GBC) is an aggressive tumour that is usually diagnosed at advanced stages and is characterised by a poor prognosis. Using public data of normal human tissues, we found that mRNA and protein levels of mucin 5B (MUC5B) and carbonic anhydrase 9 (CA9) were highly increased in gallbladder tissues. In addition, previous evidence has shown that claudin 18 (CLDN18) protein expression is higher in GBC. The aim of this study was to perform an analysis of these cell surface proteins during the histological progression of GBC in order to identify their theranostic potential. METHODS AND RESULTS: MUC5B expression, CA9 expression and CLDN18 expression were examined by immunohistochemistry in a series of 179 chronic cholecystitis (including 16 metaplastic tissues), 15 dysplasia and 217 GBC samples by the use of tissue microarray analysis. A composite staining score was calculated from staining intensity and percentage of positive cells. Immunohistochemical analysis showed high expression of MUC5B and CA9 among normal epithelium, metaplastic tissues, and dysplastic tissues. However, expression of both proteins was observed in roughly 50% of GBC samples. In contrast, CLDN18 was absent in normal epithelium, but its expression was higher in metaplastic cells. Among GBC cases, approximately half showed high CLDN18 expression. No associations were found between MUC5B, CA9 and CLDN18 expression and any clinicopathological features. CONCLUSIONS: CLDN18 is a new metaplasia marker in gallbladder tissues, and is conserved in approximately half of GBC cases. MUC5B and CA9 are highly conserved during GBC histological progression. The three markers are potential theranostic markers, in particular CA9 and CLDN18, for which there are already targeted therapies available.

ADAR1-mediated RNA-editing of 3'UTRs in breast cancer.

BACKGROUND: Whole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA (ADAR) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed. RESULTS: We characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3'UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3'UTRs. Interestingly, editing was particularly increased in the 3'UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1). CONCLUSIONS: Altogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.

The low-abundance transcriptome reveals novel biomarkers, specific intracellular pathways and targetable genes associated with advanced gastric cancer.

Studies on the low-abundance transcriptome are of paramount importance for identifying the intimate mechanisms of tumor progression that can lead to novel therapies. The aim of the present study was to identify novel markers and targetable genes and pathways in advanced human gastric cancer through analyses of the low-abundance transcriptome. The procedure involved an initial subtractive hybridization step, followed by global gene expression analysis using microarrays. We observed profound differences, both at the single gene and gene ontology levels, between the low-abundance transcriptome and the whole transcriptome. Analysis of the low-abundance transcriptome led to the identification and validation by tissue microarrays of novel biomarkers, such as LAMA3 and TTN; moreover, we identified cancer type-specific intracellular pathways and targetable genes, such as IRS2, IL17, IFNγ, VEGF-C, WISP1, FZD5 and CTBP1 that were not detectable by whole transcriptome analyses. We also demonstrated that knocking down the expression of CTBP1 sensitized gastric cancer cells to mainstay chemotherapeutic drugs. We conclude that the analysis of the low-abundance transcriptome provides useful insights into the molecular basis and treatment of cancer.

A Comprehensive Analysis of the Effect of A>I(G) RNA-Editing Sites on Genotoxic Drug Response and Progression in Breast Cancer.

Dysregulated A>I(G) RNA editing, which is mainly catalyzed by ADAR1 and is a type of post-transcriptional modification, has been linked to cancer. A low response to therapy in breast cancer (BC) is a significant contributor to mortality. However, it remains unclear if there is an association between A>I(G) RNA-edited sites and sensitivity to genotoxic drugs. To address this issue, we employed a stringent bioinformatics approach to identify differentially RNA-edited sites (DESs) associated with low or high sensitivity (FDR 0.1, log2 fold change 2.5) according to the IC50 of PARP inhibitors, anthracyclines, and alkylating agents using WGS/RNA-seq data in BC cell lines. We then validated these findings in patients with basal subtype BC. These DESs are mainly located in non-coding regions, but a lesser proportion in coding regions showed predicted deleterious consequences. Notably, some of these DESs are previously reported as oncogenic variants, and in genes related to DNA damage repair, drug metabolism, gene regulation, the cell cycle, and immune response. In patients with BC, we uncovered DESs predominantly in immune response genes, and a subset with a significant association (log-rank test p < 0.05) between RNA editing level in LSR, SMPDL3B, HTRA4, and LL22NC03-80A10.6 genes, and progression-free survival. Our findings provide a landscape of RNA-edited sites that may be involved in drug response mechanisms, highlighting the value of A>I(G) RNA editing in clinical outcomes for BC.

Detection of transcriptome-wide microRNA-target interactions in single cells with agoTRIBE.

MicroRNAs (miRNAs) exert their gene regulatory effects on numerous biological processes based on their selection of target transcripts. Current experimental methods available to identify miRNA targets are laborious and require millions of cells. Here we have overcome these limitations by fusing the miRNA effector protein Argonaute2 to the RNA editing domain of ADAR2, allowing the detection of miRNA targets transcriptome-wide in single cells. miRNAs guide the fusion protein to their natural target transcripts, causing them to undergo A>I editing, which can be detected by sensitive single-cell RNA sequencing. We show that agoTRIBE identifies functional miRNA targets, which are supported by evolutionary sequence conservation. In one application of the method we study microRNA interactions in single cells and identify substantial differential targeting across the cell cycle. AgoTRIBE also provides transcriptome-wide measurements of RNA abundance and allows the deconvolution of miRNA targeting in complex tissues at the single-cell level.

Increase in ADAR1p110 activates the canonical Wnt signaling pathway associated with aggressive phenotype in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) represents a challenge in the search for new therapeutic targets. TNBCs are aggressive and generate resistance to chemotherapy. Tumors of TNBC patients with poor prognosis present a high level of adenosine deaminase acting on RNA1 (ADAR1). We explore the connection of ADAR1 with the canonical Wnt signaling pathway and the effect of modulation of its expression in TNBC. Expression data from cell line sequencing (DepMap) and TCGA samples were downloaded and analyzed. We lentivirally generated an MDA-MB-231 breast cancer cell line that overexpress (OE) ADAR1p110 or an ADAR knockdown. Abundance of different proteins related to Wnt/ß-catenin pathway and activity of nuclear ß-catenin were analyzed by Western blot and luciferase TOP/FOP reporter assay, respectively. Cell invasion was analyzed by matrigel assay. In mice, we study the behavior of tumors generated from ADAR1p110 (OE) cells and tumor vascularization immunostaining were analyzed. ADAR1 connects to the canonical Wnt pathway in TNBC. ADAR1p110 overexpression decreased GSK-3ß, while increasing active ß-catenin. It also increased the activity of nuclear ß-catenin and increased its target levels. ADAR1 knockdown has the opposite effect. MDA-MB-231 ADAR1 (OE) cells showed increased capacity of invasion. Subsequently, we observed that tumors derived from ADAR1p110 (OE) cells showed increased invasion towards the epithelium, and increased levels of Survivin and CD-31 expressed in vascular endothelial cells. These results indicate that ADAR1 overexpression alters the expression of some key components of the canonical Wnt pathway, favoring invasion and neovascularization, possibly through activation of the ß-catenin, which suggests an unknown role of ADAR1p110 in aggressiveness of TNBC tumors.

ADAR1 Transcriptome editing promotes breast cancer progression through the regulation of cell cycle and DNA damage response.

RNA editing has emerged as a novel mechanism in cancer progression. The double stranded RNA-specific adenosine deaminase (ADAR) modifies the expression of an important proportion of genes involved in cell cycle control, DNA damage response (DDR) and transcriptional processing, suggesting an important role of ADAR in transcriptome regulation. Despite the phenotypic implications of ADAR deregulation in several cancer models, the role of ADAR on DDR and proliferation in breast cancer has not been fully addressed. Here, we show that ADAR expression correlates significantly with clinical outcomes and DDR, cell cycle and proliferation mRNAs of previously reported edited transcripts in breast cancer patients. ADAR's knock-down in a breast cancer cell line produces stability changes of mRNAs involved in DDR and DNA replication. Breast cancer cells with reduced levels of ADAR show a decreased viability and an increase in apoptosis, displaying a significant decrease of their DDR activation, compared to control cells. These results suggest that ADAR plays an important role in breast cancer progression through the regulation of mRNA stability and expression of those genes involved in proliferation and DDR impacting the viability of breast cancer cells.

Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response.

The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.

TRPM4 regulates Akt/GSK3-ß activity and enhances ß-catenin signaling and cell proliferation in prostate cancer cells.

Increased expression of the TRPM4 channel has been reported to be associated with the progression of prostate cancer. However, the molecular mechanism underlying its effect remains unknown. This work found that decreasing TRPM4 levels leads to the reduced proliferation of PC3 cells. This effect was associated with a decrease in total ß-catenin protein levels and its nuclear localization, and a significant reduction in Tcf/Lef transcriptional activity. Moreover, TRPM4 silencing increases the Ser33/Ser37/Thr41 ß-catenin phosphorylated population and reduces the phosphorylation of GSK-3ß at Ser9, suggesting an increase in ß-catenin degradation as the underlying mechanism. Conversely, TRPM4 overexpression in LNCaP cells increases the Ser9 inhibitory phosphorylation of GSK-3ß and the total levels of ß-catenin and its nonphosphorylated form. Finally, PC3 cells with reduced levels of TRPM4 showed a decrease in basal and stimulated phosphoactivation of Akt1, which is likely responsible for the decrease in GSK-3ß activity in these cells. Our results also suggest that the effect of TRPM4 on Akt1 is probably mediated by an alteration in the calcium/calmodulin-EGFR axis, linking TRPM4 activity with the observed effects in ß-catenin-related signaling pathways. These results suggest a role for TRPM4 channels in ß-catenin oncogene signaling and underlying mechanisms, highlighting this ion channel as a new potential target for future therapies in prostate cancer.

ADAR1-mediated RNA-editing of 3'UTRs in breast cancer

BACKGROUND: Whole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA ( ADAR ) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed. RESULTS: We characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3'UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3'UTRs. Interestingly, editing was particularly increased in the 3'UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1). CONCLUSIONS: Altogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.