Extracellular vesicles produced by irradiated endothelial or Glioblastoma stem cells promote tumor growth and vascularization modulating tumor microenvironment.

BACKGROUND: Glioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment. METHODS: In this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays. RESULTS: We confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration. CONCLUSION: In this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.

Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1.

Glioblastoma (GBM) is the most aggressive and prevalent form of a human brain tumor in adults. Several data have demonstrated the implication of microRNAs (miRNAs) in tumorigenicity of GBM stem-like cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. The current study aimed at investigating the function of miR-370-3p in glioma progression, as aberrant expression of miR-370-3p, is involved in various human cancers, including glioma. Analyzing our collection of GBM samples and patient-derived GSC lines, we found the expression of miR-370-3p significantly downregulated compared to normal brain tissues and normal neural stem cells. Restoration of miR-370-3p expression in GSCs significantly decreased proliferation, migration, and clonogenic abilities of GSCs, in vitro, and tumor growth in vivo. Gene expression analysis performed on miR-370-3p transduced GSCs, identified several transcripts involved in Epithelial to Mesenchymal Transition (EMT), and Hypoxia signaling pathways. Among the genes downregulated by the restored expression of miR-370-3p, we found the EMT-inducer high-mobility group AT-hook 2 (HMGA2), the master transcriptional regulator of the adaptive response to hypoxia, Hypoxia-inducible factor (HIF)1A, and the long non-coding RNAs (lncRNAs) Nuclear Enriched Abundant Transcript (NEAT)1. NEAT1 acts as an oncogene in a series of human cancers including gliomas, where it is regulated by the Epidermal Growth Factor Receptor (EGFR) pathways, and contributes to tumor growth and invasion. Noteworthy, the expression levels of miR-370-3p and NEAT1 were inversely related in both GBM tumor specimens and GSCs, and a dual-luciferase reporter assay proved the direct binding between miR-370-3p and the lncRNAs NEAT1. Our results identify a critical role of miR-370-3p in the regulation of GBM development, indicating that miR-370-3p acts as a tumor-suppressor factor inhibiting glioma cell growth, migration and invasion by targeting the lncRNAs NEAT1, HMGA2, and HIF1A, thus, providing a potential candidate for GBM patient treatment.

Critical role of IRF-8 in negative regulation of TLR3 expression by Src homology 2 domain-containing protein tyrosine phosphatase-2 activity in human myeloid dendritic cells.

Despite extensive studies that unraveled ligands and signal transduction pathways triggered by TLRs, little is known about the regulation of TLR gene expression. TLR3 plays a crucial role in the recognition of viral pathogens and induction of immune responses by myeloid DCs. IFN regulatory factor (IRF)-8, a member of the IRF family, is a transcriptional regulator that plays essential roles in the development and function of myeloid lineage, affecting different subsets of myeloid DCs. In this study, we show that IRF-8 negatively controls TLR3 gene expression by suppressing IRF-1- and/or polyinosinic-polycytidylic acid-stimulated TLR3 expression in primary human monocyte-derived DCs (MDDCs). MDDCs expressed TLR3 increasingly during their differentiation from monocytes to DCs with a peak at day 5, when TLR3 expression was further enhanced upon stimulation with polyinosinic-polycytidylic acid and then was promptly downregulated. We found that both IRF-1 and IRF-8 bind the human TLR3 promoter during MDDC differentiation in vitro and in vivo but with different kinetic and functional effects. We demonstrate that IRF-8-induced repression of TLR3 is specifically mediated by ligand-activated Src homology 2 domain-containing protein tyrosine phosphatase association. Indeed, Src homology 2 domain-containing protein tyrosine phosphatase-dephosphorylated IRF-8 bound to the human TLR3 promoter competing with IRF-1 and quashing its activity by recruitment of histone deacetylase 3. Our findings identify IRF-8 as a key player in the control of intracellular viral dsRNA-induced responses and highlight a new mechanism for negative regulation of TLR3 expression that can be exploited to block excessive TLR activation.

MiR-378a-3p Acts as a Tumor Suppressor in Colorectal Cancer Stem-Like Cells and Affects the Expression of MALAT1 and NEAT1 lncRNAs.

MiR-378a-3p plays a critical role in carcinogenesis acting as a tumor suppressor, promoting apoptosis and cell cycle arrest and reducing invasion and drug resistance in several human cancers, including colorectal cancer (CRC), where its expression is significantly associated with histological classification and prognosis. In this study, we investigated the biological and cellular processes affected by miR-378a-3p in the context of CRC carcinogenesis. In agreement with the literature, miR-378a-3p is downregulated in our cohort of CRC patients as well as, in 15 patient-derived colorectal cancer stem-like cell (CRC-SC) lines and 8 CRC cell lines, compared to normal mucosae. Restoration of miR-378a-3p restrains tumorigenic properties of CRC and CRC-SC lines, as well as, significantly reduces tumor growth in two CRC-SC xenograft mouse models. We reported that miR-378a-3p modulates the expression of the lncRNAs MALAT1 and NEAT1. Their expression is inversely correlated with that of miR-378a-3p in patient-derived CRC-SC lines. Silencing of miR-378a-3p targets, MALAT1 and NEAT1, significantly impairs tumorigenic properties of CRC-SCs, supporting the critical role of miR-378a-3p in CRC carcinogenesis as a tumor-suppressor factor by establishing a finely tuned crosstalk with lncRNAs MALAT1 and NEAT1.

IFN regulatory factor-1 negatively regulates CD4+ CD25+ regulatory T cell differentiation by repressing Foxp3 expression.

Regulatory T (Treg) cells are critical in inducing and maintaining tolerance. Despite progress in understanding the basis of immune tolerance, mechanisms and molecules involved in the generation of Treg cells remain poorly understood. IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor implicated in the regulation of various immune processes. In this study, we report that IRF-1 negatively regulates CD4(+)CD25(+) Treg cell development and function by specifically repressing Foxp3 expression. IRF-1-deficient (IRF-1(-/-)) mice showed a selective and marked increase of highly activated and differentiated CD4(+)CD25(+)Foxp3(+) Treg cells in thymus and in all peripheral lymphoid organs. Furthermore, IRF-1(-/-) CD4(+)CD25(-) T cells showed extremely high bent to differentiate into CD4(+)CD25(+)Foxp3(+) Treg cells, whereas restoring IRF-1 expression in IRF-1(-/-) CD4(+)CD25(-) T cells impaired their differentiation into CD25(+)Foxp3(+) cells. Functionally, both isolated and TGF-beta-induced CD4(+)CD25(+) Treg cells from IRF-1(-/-) mice exhibited more increased suppressive activity than wild-type Treg cells. Such phenotype and functional characteristics were explained at a mechanistic level by the finding that IRF-1 binds a highly conserved IRF consensus element sequence (IRF-E) in the foxp3 gene promoter in vivo and negatively regulates its transcriptional activity. We conclude that IRF-1 is a key negative regulator of CD4(+)CD25(+) Treg cells through direct repression of Foxp3 expression.

Deregulated expression of the imprinted DLK1-DIO3 region in glioblastoma stemlike cells: tumor suppressor role of lncRNA MEG3.

BACKGROUND: Glioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 gene‒type III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis. METHODS: Real-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3). RESULTS: Loss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan-Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT). CONCLUSION: In GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies.

IRF-1 is required for full NF-kappaB transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer.

Human immunodeficiency virus type 1 (HIV-1) gene expression is controlled by a complex interplay between viral and host factors. We have previously shown that interferon-regulatory factor 1 (IRF-1) is stimulated early after HIV-1 infection and regulates promoter transcriptional activity even in the absence of the viral transactivator Tat. In this work we demonstrate that IRF-1 is also required for full NF-kappaB transcriptional activity. We provide evidence that IRF-1 and NF-kappaB form a functional complex at the long terminal repeat (LTR) kappaB sites, which is abolished by specific mutations in the two adjacent kappaB sites in the enhancer region. Silencing IRF-1 with small interfering RNA resulted in impaired NF-kappaB-mediated transcriptional activity and in repressed HIV-1 transcription early in de novo-infected T cells. These data indicate that in early phases of HIV-1 infection or during virus reactivation from latency, when the viral transactivator is absent or present at very low levels, IRF-1 is an additional component of the p50/p65 heterodimer binding the LTR enhancer, absolutely required for efficient HIV-1 replication.

Intracellular HIV-1 Tat protein represses constitutive LMP2 transcription increasing proteasome activity by interfering with the binding of IRF-1 to STAT1.

The Tat protein is the transcriptional activator of HIV-1 gene expression, which is not only essential for viral replication, but also important in the complex HIV-induced pathogenesis of AIDS, as both an intracellular and an extracellular released protein. Accordingly, Tat is able to profoundly affect cellular gene expression, regulating several cellular functions, also in non-infected cells. We showed recently that Tat induces modification of immunoproteasomes in that it up-regulates LMP7 (low-molecular-mass polypeptide 7) and MECL1 (multicatalytic endopeptidase complex-like 1) subunits and down-modulates the LMP2 subunit, resulting in a change in the generation and presentation of epitopes in the context of MHC class I. In particular, Tat increases presentation of subdominant and cryptic epitopes. In the present study, we investigated the molecular mechanism responsible for the Tat-induced LMP2 down-regulation and show that intracellular Tat represses transcription of the LMP2 gene by competing with STAT1 (signal transducer and activator of transcription 1) for binding to IRF-1 (interferon-regulatory factor-1) on the overlapping ICS-2 (interferon consensus sequence-2)-GAS (gamma-interferon-activated sequence) present in the LMP2 promoter. This element is constitutively occupied in vivo by the unphosphorylated STAT1-IRF-1 complex, which is responsible for the basal transcription of the gene. Sequestration of IRF-1 by intracellular Tat impairs the formation of the complex resulting in lower LMP2 gene transcription and LMP2 protein expression, which is associated with increased proteolytic activity. On the other hand, extracellular Tat induces the expression of LMP2. These effects of Tat provide another effective mechanism by which HIV-1 affects antigen presentation in the context of the MHC class I complex and may have important implications in the use of Tat for vaccination strategies.

A three-microRNA signature identifies two subtypes of glioblastoma patients with different clinical outcomes.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults, characterized by aggressive growth, limited response to therapy, and inexorable recurrence. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant patient stratification systems. Analyzing a collection of patient-derived GBM stem-like cells (GSCs) by gene expression profiling, nuclear magnetic resonance spectroscopy, and signal transduction pathway activation, we identified two GSC clusters characterized by different clinical features. Due to the widely documented role played by microRNAs (miRNAs) in the tumorigenesis process, in this study we explored whether these two GBM patient subtypes could also be discriminated by different miRNA signatures. Global miRNA expression pattern was analyzed by oblique principal component analysis and principal component analysis. By a combined inferential strategy on PCA results, we identified a reduced set of three miRNAs - miR-23a, miR-27a, and miR-9* (miR-9-3p) - able to discriminate the proneural- and mesenchymal-like GSC phenotypes as well as mesenchymal and proneural subtypes of primary GBM included in The Cancer Genome Atlas (TCGA) data set. Kaplan-Meier analysis showed a significant correlation between the selected miRNAs and overall survival in 429 GBM specimens from TCGA-identifying patients who had an unfavorable outcome. The survival prognostic capability of the three-miRNA signatures could have important implications for the understanding of the biology of GBM subtypes and could be useful in patient stratification to facilitate interpretation of results from clinical trials.

PML-RAR alpha induces the downmodulation of HHEX: a key event responsible for the induction of an angiogenetic response.

BACKGROUND: Recent studies indicate that angiogenesis is important in the pathogenesis of acute myeloid leukemias (AMLs). Among the various AMLs, the bone marrow angiogenetic response is particularly pronounced in acute promyelocytic leukemia (APL). However, the molecular mechanisms responsible for this angiogenetic response are largely unknown. In the present study, we have explored the role of HHEX, a homeodomain transcription factor, as a possible mediator of the pro-angiogenetic response observed in APL. This transcription factor seems to represent an ideal candidate for this biologic function because it is targeted by PML-RARα, is capable of interaction with PML and PML-RARα, and acts as a regulator of the angiogenetic response. METHODS: We used various cellular systems of APL, including primary APL cells and leukemic cells engineered to express PML-RARα, to explore the role of the PML-RARα fusion protein on HHEX expression. Molecular and biochemical techniques have been used to investigate the mechanisms through which PML-RARα downmodulates HHEX and the functional consequences of this downmodulation at the level of the expression of various angiogenetic genes, cell proliferation and differentiation. RESULTS: Our results show that HHEX expression is clearly downmodulated in APL and that this effect is directly mediated by a repressive targeting of the HHEX gene promoter by PML-RARα. Studies carried out in primary APL cells and in a cell line model of APL with inducible PML-RARα expression directly support the view that this fusion protein through HHEX downmodulation stimulates the expression of various genes involved in angiogenesis and inhibits cell differentiation. CONCLUSIONS: Our data suggest that HHEX downmodulation by PML-RARα is a key event during APL pathogenesis.

Role of Ets-1 in transcriptional regulation of transferrin receptor and erythroid differentiation.

High expression of transferrin receptor (TfR) on the membrane of erythroid cells accounts for the high level of iron required to sustain heme synthesis. Several studies indicate that during erythroid differentiation TfR expression is highly dependent on transcriptional regulation. In this study we characterized the minimal region able to confer transcriptional regulation during erythroid differentiation in Friend leukemia cells (FLC). This region of 120 bp, upstream the transcription start site, contains an overlapping consensus recognition sequence for AP1/CREB/ATF transcription factors and for proteins of the Ets family and a GC rich region. Here, we report that both the Ets and the Ap1/CRE like sites are essential for promoter activity during erythroid differentiation. We showed that Ets-1 binds to the EBS-TfR and its binding activity decreases in FLC induced to differentiate and during normal erythroid differentiation. Consistent with this, FLC constitutively expressing Ets-1 show a decrease in TfR gene expression, globin mRNA and hemoglobin synthesis. We conclude that Ets-1 binding activity is modulated during erythroid maturation and that a deregulated expression of this transcription factor interferes with terminal erythroid differentiation.

miR-135b suppresses tumorigenesis in glioblastoma stem-like cells impairing proliferation, migration and self-renewal.

Glioblastoma multiforme (GBM) is the most common and fatal malignant adult primary brain tumor. Currently, the overall prognosis for GBM patients remains poor despite advances in neurosurgery and adjuvant treatments. MicroRNAs (miRNAs) contribute to the pathogenesis of various types of tumor, including GBM. In this study we analyzed the expression of a panel of miRNAs, which are known to be differentially expressed by the brain and GBM tumor, in a collection of patient-derived GBM stem-like cells (GSCs). Notably, the average expression level of miR-135b, was the most downregulated compared to its normal counterpart, suggesting a potential role as anti-oncogene.Restoration of miR-135b in GSCs significantly decreased proliferation, migration and clonogenic abilities. More importantly, miR-135b restoration was able to significantly reduce brain infiltration in mouse models of GBM obtained by intracerebral injection of GSC lines. We identified ADAM12 and confirmed SMAD5 and GSK3ß as miR-135b targets and potential mediators of its effects. The whole transcriptome analysis ascertained that the expression of miR-135b downmodulated additional genes driving key pathways in GBM survival and infiltration capabilities.Our results identify a critical role of miR-135b in the regulation of GBM development, suggesting that miR-135b might act as a tumor-suppressor factor and thus providing a potential candidate for the treatment of GBM patients.

Role of Ets-1 in erythroid differentiation.

Members of the Ets gene family are known to be expressed in the hematopoietic tissue and some of them play a pivotal role in normal hematopoietic cell development. Ets-1 gene expression was analyzed in Friend Leukemia Cells (FLC) induced to erythroid differentiation by DMSO. We show that the level of Ets-1 protein and its binding activity decreases in FLC along erythroid differentiation of primary human progenitors. The same behavior was observed during normal erythroid differentiation. Moreover, FLC constitutively expressing Ets-1 show a decrease in TfR gene expression, globin mRNA and hemoglobin synthesis. These data indicate that a decrease in Ets-1 binding activity is required for a normal erythroid maturation and that a deregulated expression of this transcription factor may interfere with terminal erythroid differentiation.