Long Noncoding RNAs and Cancer Stem Cells: Dangerous Liaisons Managing Cancer.

Decades of research have investigated the mechanisms that lead to the origin of cancer, striving to identify tumor-initiating cells. These cells, also known as cancer stem cells, are characterized by the ability to self-renew, to give rise to differentiated tumor populations, and on a larger scale, are deemed responsible not only for tumor initiation but also for recurrent tumors, often resistant to chemotherapy and radiotherapy. Long noncoding RNAs are RNA molecules longer than 200 nt, lacking the ability to code for proteins, with recognized roles as fine regulators of gene expression. They can exert these functions through a variety of mechanisms, acting at almost all steps of gene expression, from modulation of the epigenetic state of chromatin to modulation of protein stability. In all cases, lncRNAs do not work alone, but they always interact with other RNA molecules, either coding or non-coding, or with protein factors. In this review, we summarize the latest results obtained about the involvement of lncRNAs in the initiating cells of several types of tumors, and highlight the different mechanisms through which they work, while discussing how the modulation of a lncRNA can affect several aspects of tumor onset and progression.

Glioblastoma stem cells express non-canonical proteins and exclusive mesenchymal-like or non-mesenchymal-like protein signatures.

Glioblastoma (GBM) cancer stem cells (GSCs) contribute to GBM's origin, recurrence, and resistance to treatment. However, the understanding of how mRNA expression patterns of GBM subtypes are reflected at global proteome level in GSCs is limited. To characterize protein expression in GSCs, we performed in-depth proteogenomic analysis of patient-derived GSCs by RNA-sequencing and mass-spectrometry. We quantified > 10 000 proteins in two independent GSC panels and propose a GSC-associated proteomic signature characterizing two distinct phenotypic conditions; one defined by proteins upregulated in proneural and classical GSCs (GPC-like), and another by proteins upregulated in mesenchymal GSCs (GM-like). The GM-like protein set in GBM tissue was associated with necrosis, recurrence, and worse overall survival. Through proteogenomics, we discovered 252 non-canonical peptides in the GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered non-protein-coding, including variants of the heterogeneous ribonucleoproteins implicated in RNA splicing. In summary, GSCs express two protein sets that have an inverse association with clinical outcomes in GBM. The discovery of non-canonical protein sequences questions existing gene models and pinpoints new protein targets for research in GBM.

MEOX2 Regulates the Growth and Survival of Glioblastoma Stem Cells by Modulating Genes of the Glycolytic Pathway and Response to Hypoxia.

The most widely accepted hypothesis for the development of glioblastoma suggests that glioblastoma stem-like cells (GSCs) are crucially involved in tumor initiation and recurrence as well as in the occurrence of chemo- and radio-resistance. Mesenchyme homeobox 2 (MEOX2) is a transcription factor overexpressed in glioblastoma, whose expression is negatively correlated with patient survival. Starting from our observation that MEOX2 expression is strongly enhanced in six GSC lines, we performed shRNA-mediated knock-down experiments in two different GSC lines and found that MEOX2 depletion resulted in the inhibition of cell growth and sphere-forming ability and an increase in apoptotic cell death. By a deep transcriptome analysis, we identified a core group of genes modulated in response to MEOX2 knock-down. Among these genes, the repressed ones are largely enriched in genes involved in the hypoxic response and glycolytic pathway, two strictly related pathways that contribute to the resistance of high-grade gliomas to therapies. An in silico study of the regulatory regions of genes differentially expressed by MEOX2 knock-down revealed that they mainly consisted of GC-rich regions enriched for Sp1 and Klf4 binding motifs, two main regulators of metabolism in glioblastoma. Our results show, for the first time, the involvement of MEOX2 in the regulation of genes of GSC metabolism, which is essential for the survival and growth of these cells.

The RNA editing enzyme ADAR2 restricts L1 mobility.

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in double-stranded RNAs (RNA editing A-to-I). ADAR1 and ADAR2 were previously reported as HIV-1 proviral factors. The aim of this study was to investigate the composition of the ADAR2 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 10 non-ribosomal ADAR2-interacting factors. A significant fraction of these proteins was previously found associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons. Considering that we previously demonstrated that ADAR1 is an inhibitor of LINE-1 retrotransposon activity, we investigated whether also ADAR2 played a similar function. To reach this goal, we performed specific cell culture retrotransposition assays in cells overexpressing or ablated for ADAR2. These experiments unveil a novel function of ADAR2 as suppressor of L1 retrotransposition. Furthermore, we showed that ADAR2 binds the basal L1 RNP complex.Overall, these data support the role of ADAR2 as regulator of L1 life cycle.

RNA Editing in Interferonopathies.

The type I interferonopathies comprise a heterogenous group of monogenic diseases associated with a constitutive activation of type I interferon signaling.The elucidation of the genetic causes of this group of diseases revealed an alteration of nucleic acid processing and signaling.ADAR1 is among the genes found mutated in patients with this type of disorders.This enzyme catalyzes the hydrolytic deamination of adenosines in inosines within a double-stranded RNA target (RNA editing of A to I). This RNA modification is widespread in human cells and deregulated in a variety of human diseases, ranging from cancers to neurological abnormalities.In this review, we briefly summarize the knowledge about the RNA editing alterations occurring in patients with mutations in ADAR1 gene and how these alterations might cause the inappropriate IFN activation.

Insights into the Regulatory Role of m6A Epitranscriptome in Glioblastoma.

N6-methyladenosine (m6A) is one of the most widespread and abundant internal messenger RNA modifications found in eukaryotes. Emerging evidence suggests that this modification is strongly linked to the activation and inhibition of cancer pathways and is associated with prognostically significant tumour subtypes. The present review describes the dynamic nature of m6A regulator enzymes, as methyltransferases, demethylases and m6A binding proteins, and points out thevalue of the balance among these proteins in regulating gene expression, cell metabolism and cancer development. The main focus of this review is on the roles of m6A modification in glioblastoma, the most aggressive and invariably lethal brain tumour. Although the study of m6A in glioblastoma is a young one, and papers in this field can yield divergent conclusions, the results collected so far clearly demonstrate that modulation of mRNA m6A levels impacts multiple aspects of this tumour, including growth, glioma stem cells self-renewal, and tumorigenesis, suggesting that mRNA m6A modification may serve as a promising target for glioblastoma therapy. We also present recent data about another type of epitranscriptomic modification, the methylation of cytosine at a specific site of 28S rRNA, as it was recently shown to affect the biology of glioma cells, with high potential of clinical implications.

The Expression of the Chemokine CXCL14 Correlates with Several Aggressive Aspects of Glioblastoma and Promotes Key Properties of Glioblastoma Cells.

Glioblastoma (GBM) is a primary brain tumor whose prognosis is inevitably dismal, leading patients to death in about 15 months from diagnosis. Tumor cells in the mass of the neoplasm are in continuous exchange with cells of the stromal microenvironment, through the production of soluble molecules, among which chemokines play prominent roles. CXCL14 is a chemokine with a pro-tumor role in breast and prostate carcinoma, where it is secreted by cancer associated fibroblasts, and contributes to tumor growth and invasion. We previously observed that CXCL14 expression is higher in GBM tissues than in healthy white matter. Here, we study the effects of exogenously supplemented CXCL14 on key tumorigenic properties of human GBM cell lines. We show that CXCL14 enhances the migration ability and the proliferation of U87MG and LN229 GBM cell lines. None of these effects was affected by the use of AMD3100, an inhibitor of CXCR4 receptor, suggesting that the observed CXCL14 effects are not mediated by this receptor. We also provide evidence that CXCL14 enhances the sphere-forming ability of glioblastoma stem cells, considered the initiating cells, and is responsible for tumor onset, growth and recurrence. In support of our in vitro results, we present data from several GBM expression datasets, demonstrating that CXCL14 expression is inversely correlated with overall survival, that it is enriched at the leading edge of the tumors and in infiltrating tumor areas, and it characterizes mesenchymal and NON G-CIMP tumors, known to have a particularly bad prognosis. Overall, our results point to CXCL14 as a protumorigenic chemokine in GBM.

Dual regulation of L-selectin (CD62L) by HIV-1: Enhanced expression by Vpr in contrast with cell-surface down-modulation by Nef and Vpu.

The HIV-1 accessory protein Vpr displays various activities that can favor viral replication such as G2 cell cycle arrest. Vpr also modulates host gene expression, although this property is poorly characterized. Here, we investigated the effect of Vpr on L-selectin (CD62L), which crucially controls leukocytes circulation and generation of immune responses against pathogens. We report that Vpr up-regulates CD62L mRNA level when individually expressed in Jurkat T cells as well as during HIV-1 infection of primary CD4+ T cells. Vpr mutant analysis and use of inhibitors suggest that the effect of Vpr on CD62L occurs independently of G2 arrest but requires activation of the ATR kinase. Yet, induction of CD62L expression by Vpr is contrasted by down-regulation of CD62L protein by Nef that, together with Vpu, induces a net reduction of cell-surface CD62L on HIV-1-infected cells, which may impact viral spread and evasion of immune responses.

The lncRNA H19 positively affects the tumorigenic properties of glioblastoma cells and contributes to NKD1 repression through the recruitment of EZH2 on its promoter.

The still largely obscure molecular events in the glioblastoma oncogenesis, a primary brain tumor characterized by an inevitably dismal prognosis, impel for investigation. The importance of Long noncoding RNAs as regulators of gene expression has recently become evident. Among them, H19 has a recognized oncogenic role in several types of human tumors and was shown to correlate to some oncogenic aspects of glioblastoma cells. Here we, hypothesyze that in glioblastoma H19 exerts its function through the interaction with the catalytic subunit of the PRC2 complex, EZH2. By employing a factor analysis on a SAGE dataset of 12 glioblastoma samples, we show that H19 expression in glioblastoma tissues correlates with that of several genes involved in glioblastoma growth and progression. H19 knock-down reduces viability, migration and invasiveness of two distinct human glioblastoma cell lines. Most importantly, we provide a mechanistic perspective about the role of H19 in glioblastoma cells, by showing that its expression is inversely linked to that of NKD1, a negative regulator of Wnt pathway, suggesting that H19 might regulate NKD1 transcription via EZH2-induced H3K27 trimethylation of its promoter. Indeed, we showed that H19 binds EZH2 in glioblastoma cells, and that EZH2 binding to NKD1 and other promoters is impaired by H19 silencing. In this work we describe H19 as part of an epigenetic modulation program executed by EZH2, that results in the repression of Nkd1. We believe that our results can provide a new piece to the complex puzzle of H19 function in glioblastoma.

Post-transcriptional regulation of LINE-1 retrotransposition by AID/APOBEC and ADAR deaminases.

Long interspersed element-1 (LINE-1 or L1) retrotransposons represent the only functional family of autonomous transposable elements in humans and formed 17% of our genome. Even though most of the human L1 sequences are inactive, a limited number of copies per individual retain the ability to mobilize by a process termed retrotransposition. The ongoing L1 retrotransposition may result in insertional mutagenesis that could lead to negative consequences such as genetic disease and cancer. For this reason, cells have evolved several mechanisms of defense to restrict L1 activity. Among them, a critical role for cellular deaminases [activation-induced deaminase (AID)/apolipoprotein B mRNA-editing catalytic polypeptide-like (APOBEC) and adenosine deaminases that act on RNA (ADAR) enzymes] has emerged. The majority of the AID/APOBEC family of proteins are responsible for the deamination of cytosine to uracil (C-to-U editing) within DNA and RNA targets. The ADARs convert adenosine bases to inosines (A-to-I editing) within double-stranded RNA (dsRNA) targets. This review will discuss the current understanding of the regulation of LINE-1 retrotransposition mediated by these enzymes.

MicroRNAs as Multifaceted Players in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common and inevitably lethal primary brain tumor, with a median survival rate of only 15 months from diagnosis. The current standard treatment involves maximal surgical resection flanked by radiotherapy and chemotherapy with the alkylating agent temozolomide. However, even such aggressive treatment is never curative, and recurrent tumors always arise, commonly in more aggressive, chemo- and radio-resistant forms, leading to untreatable and deadly tumors. MicroRNAs, recognized major players in cancer, are deeply involved in GBM, as shown by more than a decade of studies. In this review, we revise the main milestones of MicroRNA studies in GBM, and the latest relevant discoveries in this field. Examples are given of MicroRNAs working as "oncomiRs" or tumor suppressors, with specific connections with GBM clinical subtypes, patients' survival, and resistance to therapies. As the interaction of GBM cells with the microenvironment was proven as a key determinant of tumor growth, the role of MicroRNAs in GBM microenvironment, tumor angiogenesis, and tumor-secreted microvesicles is also reviewed. Finally, we discuss the latest findings presenting MicroRNAs as possible therapeutic targets for GBM, or their use as circulating biomarkers in diagnosis and prognosis.

ADAR1 restricts LINE-1 retrotransposition.

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosines to inosines in double-stranded RNAs. ADAR1 was demonstrated to be functional on different viruses exerting either antiviral or proviral effects. Concerning HIV-1, several studies showed that ADAR1 favors viral replication. The aim of this study was to investigate the composition of the ADAR1 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 14 non-ribosomal ADAR1-interacting proteins, most of which are novel. A significant fraction of these proteins were previously demonstrated to be associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons that continuously re-enter host-genome.Hence, we investigated the function of ADAR1 in the regulation of L1 activity.By using different cell-culture based retrotransposition assays in HeLa cells, we demonstrated a novel function of ADAR1 as suppressor of L1 retrotransposition. Apparently, this inhibitory mechanism does not occur through ADAR1 editing activity. Furthermore, we showed that ADAR1 binds the basal L1 RNP complex. Overall, these data support the role of ADAR1 as regulator of L1 life cycle.

Resetting cancer stem cell regulatory nodes upon MYC inhibition.

MYC deregulation is common in human cancer and has a role in sustaining the aggressive cancer stem cell populations. MYC mediates a broad transcriptional response controlling normal biological programmes, but its activity is not clearly understood. We address MYC function in cancer stem cells through the inducible expression of Omomyc-a MYC-derived polypeptide interfering with MYC activity-taking as model the most lethal brain tumour, glioblastoma. Omomyc bridles the key cancer stemlike cell features and affects the tumour microenvironment, inhibiting angiogenesis. This occurs because Omomyc interferes with proper MYC localization and itself associates with the genome, with a preference for sites occupied by MYC This is accompanied by selective repression of master transcription factors for glioblastoma stemlike cell identity such as OLIG2, POU3F2, SOX2, upregulation of effectors of tumour suppression and differentiation such as ID4, MIAT, PTEN, and modulation of the expression of microRNAs that target molecules implicated in glioblastoma growth and invasion such as EGFR and ZEB1. Data support a novel view of MYC as a network stabilizer that strengthens the regulatory nodes of gene expression networks controlling cell phenotype and highlight Omomyc as model molecule for targeting cancer stem cells.

CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27(Kip1) mRNA translation.

The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via 3' untranslated regions (3'UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27(Kip1) by specifically targeting its 3'UTR, and competes with miR-221/222 binding at an overlapping site in the 3'UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27(Kip1) 3'UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27(Kip1) mRNA. This leads to higher levels of p27(Kip1) in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma.

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

BACKGROUND: ADAR 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. RESULTS: By 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. CONCLUSIONS: Our 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.

HIV-1 infection causes a down-regulation of genes involved in ribosome biogenesis.

HIV-1 preferentially infects CD4+ T cells, causing fundamental changes that eventually lead to the release of new viral particles and cell death. To investigate in detail alterations in the transcriptome of the CD4+ T cells upon viral infection, we sequenced polyadenylated RNA isolated from Jurkat cells infected or not with HIV-1. We found a marked global alteration of gene expression following infection, with an overall trend toward induction of genes, indicating widespread modification of the host biology. Annotation and pathway analysis of the most deregulated genes showed that viral infection produces a down-regulation of genes associated with the nucleolus, in particular those implicated in regulating the different steps of ribosome biogenesis, such as ribosomal RNA (rRNA) transcription, pre-rRNA processing, and ribosome maturation. The impact of HIV-1 infection on genes involved in ribosome biogenesis was further validated in primary CD4+ T cells. Moreover, we provided evidence by Northern Blot experiments, that host pre-rRNA processing in Jurkat cells might be perturbed during HIV-1 infection, thus strengthening the hypothesis of a crosstalk between nucleolar functions and viral pathogenesis.

The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222.

Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.

microRNAs and RNA-binding proteins: a complex network of interactions and reciprocal regulations in cancer.

In the last decade, an ever-growing number of connections between microRNAs (miRNAs) and RNA-binding proteins (RBPs) have uncovered a new level of complexity of gene expression regulation in cancer. In this review, we examine several aspects of the functional interactions between miRNAs and RBPs in cancer models. We will provide examples of reciprocal regulation: miRNAs regulating the expression of RBPs, or the converse, where an RNA-binding protein specifically regulates the expression of a specific miRNA, or when an RBP can exert a widespread effect on miRNAs via the modulation of a key protein for miRNA production or function. Moreover, we will focus on the ever-growing number of functional interactions that have been discovered in the last few years: RBPs that were shown to cooperate with microRNAs in the downregulation of shared target mRNAs or, on the contrary, that inhibit microRNA action, thus resulting in a protection of the specific target mRNAs. We surely need to obtain a deeper comprehension of such intricate networks to have a chance of understanding and, thus, fighting cancer.

NF-kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cells.

MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in all aspects of cell biology. They finely modulate virtually all physiological pathways in metazoans, and are deeply implicated in all main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription.