ATRA modulates the expression of alternative Transglutaminase 2 in brain tumors through interactions between regulatory factors and intronic regions

Transglutaminase 2 is multifunction enzyme encoded from a very long gene (among 40 kb) responsible to several stimuli (TNFα, TGFβ, INFγ, NF-kB, cytokines, interleukins, hormones) that rapidly induce transcription process. Consequently to strong induction, this as other genes belonging to network TGFβ/NF-kB presents pausing sites which regulate RNApol II speed during elongation. The major checkpoint is located at the beginning of the gene where the insulators RAD21 and CTCF are engaged in the chromatin remodelling in corrispondence to H3K4me3 mark and a region containing regulatory lncRNA. Notably TGM2 synthetize several altered isoforms mostly lacking 3’terminal portion that have lost cellular functions, such as signals localization and hydrolysis of GTP which affects also interaction with PLCδ1. These variants are produces by alternative transcripts using different polyadenylation or splicing sites and are associated to inflammatory processes, neurodegenerative diseases and cancer. In particular, the canonical full-length TG2 is linked to Epithelial-Mesenchymal Transition, cell survival, while the truncated TGH (the most expressed among the variants) promotes differentiation and cell death. From this point of view study TGM2 gene expression could useful to investigate the responsiveness and/or resistance to drugs. Indeed, molecules such as retinoids, as well as other anticancer compounds, change the ratio among the variants possibly modifying cellular signals to drive cancer cells to apoptosis or to therapeutic ineffectiveness. In this work we analyse the expression of the alternative transcripts of TG2 in brain tumor cell lines after modulation with all-trans retinoic acid, an agent employed in combined therapies. With the aim to investigate whether there are factors interacting with intronic sequences putatively involved in the generation of TGM2 variants we examined ChIP-seq data from ENCODE on cells untreated or treated with ATRA, highlighting those which could play regulatory roles. Behaviour of ATRA thinly modulates the levels of isoforms modifying interactions between factors and intronic DNA sequence likely interfering with RNApol II at checkpoints along the gene. These interactions influence the use of alternative polyadenylation and splicing sites, hence the production of altered isoform

Analysis of Differential Expression of TG2 Variants During Treatment with Retinoic Acid Highlights a New 3'-Terminal Transcript and Putatively Involved Proteins

In the last few years great attention has been aimed at understanding the role of alternative splicing and differential expression of variant isoforms especially as hallmarks of cancer, in cell death and in neurodegenerative processes. In this context it was reported that Transglutaminase 2 expression is associated with tumor status, progression and response to applied therapies, and that distinct isoforms of the enzyme are detected in several neurodegenerative diseases. In this work we have analyzed the modulation of TGM2 variants and other gene transcripts in hematologic and neurogenic cell lines after treatment with retinoic acid, a well known differentiating agent also used in combined therapies for glioblastoma and acute leukaemia. Our data indicate that treatment of acute progranulocytic leukaemia HL-60 cells with ATRA for variable time intervals (16-80hs) increased the expression of the truncated and alternatively-spliced variants and induced also a short 3’-terminal transcript associated with up-regulation of TGM2, along with the lncRNA included in the gene. This new 3’-terminal transcript is constituted by the sequence corresponding to the last 3 exons of the gene and is strongly modulated by ATRA in HL-60 cells. We scrutinized their expression in the early stages after treatment (0-8h) of HL-60 comparing the results with those obtained in erythroleukemia K562 cells. These latter cells have mutational inactivation of the p53 and hypomethylation of the promoter, thus supporting a basal level of transglutaminase expression and undergoing apoptosis upon administration of ATRA. In contrast to what happens in HL-60, the short 3’-terminal transcript was detected also in untreated K562 cells, and likely decreasing upon ATRA administration. We have extended the analysis to gene expression profiling for TGM2 in other cell lines responsive to retinoic acids: acute promyelocytic leukemia NB4 cells and glioblastoma GBM cells, both tumors arising from astrocytes. In both cases we observed accumulation of TGM2 transcriptional variants associated with an increase of the lncRNA and the short 3’-terminal transcript. To attempt to identify the transcriptional factors that could be involved in the differential expression of these transcripts we analyzed ChIP-seq experiments from the ENCODE database performed on neuroblastoma SK-N-SH cell line untreated and treated with retinoic acid, focusing attention on TGM2 gene promoter and intronic regions recognized by transcription or DNA-binding factors. The results paired to that differentially expressed in some of the cell lines used in this study could be helpful to shed light on transcriptional ATRA-modulation of TGM2 isoforms, where lncRNA seems to play a role as enhancer RNA. It will be interesting to further explore whether the overexpression of a specific transcript is also followed by concomitant increase of the alternative enzyme variant(s). These expression studies about TGM2 variants in tumour cells could represent a valuable tool to investigate their association with the progression and invasiveness of the disease and be also a prognostic factor for effectiveness or resistance to chemotherapeutic agents. A deeper understanding of the factors involved in gene modulation of TGM2 variants could be helpful to develop innovative approaches for gene therapy

Regulatory RNAs of the Transglutaminase 2 gene interfere with its expression in leukaemic cell lines after ATRA treatment

TGM2 is a long gene modulated by ATRA, which generates truncated variants by RNApol II pausing at checkpoints through alternative splicing and polyadenylation, along with two non-coding RNAs. One is a lncRNA transcribed from the first intron, while the other RNA, designed as LEV (Last Exons Variant), starts from intron 10 and contains the last 3 exons including the 3’UTR of the gene. The structure of the lncRNA suggests its multifunctional role, while LEV mimics exactly the last portion of the full-length TG2 transcript. RNA immunoprecipitation experiments and the use of antisense oligonucleotides demonstrate that lncRNA binds RNApol II interfering with the expression of full-length TG2 and LEV, while LEV appears associated to modulation of the altered TGH levels in mechanisms dependent on the involvement of the transcriptional factor GATA3. The functional significance of these findings will be discussed in relation with the presence of altered enzyme isoforms

miR-125b targets erythropoietin and its receptor and their expression correlates with metastatic potential and ERBB2/HER2 expression

Background The microRNA 125b is a double-faced gene expression regulator described both as a tumor suppressor gene (in solid tumors) and an oncogene (in hematologic malignancies). In human breast cancer, it is one of the most down-regulated miRNAs and is able to modulate ERBB2/3 expression. Here, we investigated its targets in breast cancer cell lines after miRNA-mimic transfection. We examined the interactions of the validated targets with ERBB2 oncogene and the correlation of miR-125b expression with clinical variables. Methods MiR-125b possible targets were identified after transfecting a miRNA-mimic in MCF7 cell line and analyzing gene expression modifications with Agilent microarrays and Sylamer bioinformatic tool. Erythropoietin (EPO) and its receptor (EPOR) were validated as targets of miR-125b by luciferase assay and their expression was assessed by RT-qPCR in 42 breast cancers and 13 normal samples. The molecular talk between EPOR and ERBB2 transcripts, through miR-125b, was explored transfecting MDA-MD-453 and MDA-MB-157 with ERBB2 RNA and using RT-qPCR. Results We identified a panel of genes down-regulated after miR-125b transfection and putative targets of miR-125b. Among them, we validated erythropoietin (EPO) and its receptor (EPOR) - frequently overexpressed in breast cancer - as true targets of miR-125b. Moreover, we explored possible correlations with clinical variables and we found a down-regulation of miR-125b in metastatic breast cancers and a significant positive correlation between EPOR and ERBB2/HER2 levels, that are both targets of miR-125b and function as competing endogenous RNAs (ceRNAs). Conclusions Taken together our results show a mechanism for EPO/EPOR and ERBB2 co-regulation in breast cancer and confirm the importance of miR-125b in controlling clinically-relevant cancer features

NAD(+) repletion with niacin counteracts cancer cachexia

Cachexia is a debilitating wasting syndrome and highly prevalent comorbidity in cancer patients. It manifests especially with energy and mitochondrial metabolism aberrations that promote tissue wasting. We recently identified nicotinamide adenine dinucleotide (NAD(+)) loss to associate with muscle mitochondrial dysfunction in cancer hosts. In this study we confirm that depletion of NAD(+) and downregulation of Nrk2, an NAD(+) biosynthetic enzyme, are common features of severe cachexia in different mouse models. Testing NAD(+) repletion therapy in cachectic mice reveals that NAD(+) precursor, vitamin B3 niacin, efficiently corrects tissue NAD(+) levels, improves mitochondrial metabolism and ameliorates cancer- and chemotherapy-induced cachexia. In a clinical setting, we show that muscle NRK2 is downregulated in cancer patients. The low expression of NRK2 correlates with metabolic abnormalities underscoring the significance of NAD(+) in the pathophysiology of human cancer cachexia. Overall, our results propose NAD(+) metabolism as a therapy target for cachectic cancer patients.The loss of nicotinamide adenine dinucleotide is reported to be associated with muscle mitochondrial dysfunction in murine cancer models. Here the authors show that niacin supplementation improves mitochondrial metabolism and reduces muscle wasting in mouse models of cachexia.Peer reviewe

Satellitome Analysis in the Southern Lapwing (<i>Vanellus chilensis</i>) Genome: Implications for SatDNA Evolution in Charadriiform Birds

Vanellus (Charadriidae; Charadriiformes) comprises around 20 species commonly referred to as lapwings. In this study, by integrating cytogenetic and genomic approaches, we assessed the satellite DNA (satDNA) composition of one typical species, Vanellus chilensis, with a highly conserved karyotype. We additionally underlined its role in the evolution, structure, and differentiation process of the present ZW sex chromosome system. Seven distinct satellite DNA families were identified within its genome, accumulating on the centromeres, microchromosomes, and the W chromosome. However, these identified satellite DNA families were not found in two other Charadriiformes members, namely Jacana jacana and Calidris canutus. The hybridization of microsatellite sequences revealed the presence of a few repetitive sequences in V. chilensis, with only two out of sixteen displaying positive hybridization signals. Overall, our results contribute to understanding the genomic organization and satDNA evolution in Charadriiform birds