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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.

Converging evidence indicates that the Fragile X Messenger Ribonucleoprotein (FMRP), which absent or mutated in Fragile X Syndrome (FXS), plays a role in many types of cancers. However, while FMRP roles in brain development and function have been extensively studied, its involvement in the biology of brain tumors remains largely unexplored. Here we show, in human glioblastoma (GBM) biopsies, that increased expression of FMRP directly correlates with a worse patient outcome. In contrast, reductions in FMRP correlate with a diminished tumor growth and proliferation of human GBM stem-like cells (GSCs) in vitro in a cell culture model and in vivo in mouse brain GSC xenografts. Consistently, increased FMRP levels promote GSC proliferation. To characterize the mechanism(s) by which FMRP regulates GSC proliferation, we performed GSC transcriptome analyses in GSCs expressing high levels of FMRP, and in these GSCs after knockdown of FMRP. We show that the WNT signalling is the most significantly enriched among the published FMRP target genes and genes involved in ASD. Consistently, we find that reductions in FMRP downregulate both the canonical WNT/β-Catenin and the non-canonical WNT-ERK1/2 signalling pathways, reducing the stability of several key transcription factors (i.e. β-Catenin, CREB and ETS1) previously implicated in the modulation of malignant features of glioma cells. Our findings support a key role for FMRP in GBM cancer progression, acting via regulation of WNT signalling.

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.

Objective Axitinib is a tyrosine kinase inhibitor characterized by a strong affinity for Vascular Endothelial Growth Factor Receptors (VEGFRs). It was approved in 2012 by Food and Drug Administration and European Medicines Agency as a second line treatment for advanced renal cell carcinoma and is currently under evaluation in clinical trial for the treatment of other cancers. Glioblastoma IDH‐wild type (GBM) is a highly malignant brain tumor characterized by diffusely infiltrative growth pattern and by a prominent neo‐angiogenesis. In GBM, axitinib has demonstrated a limited effectiveness as a monotherapy, while it was recently shown to significantly improve its efficacy in combination treatments. In preclinical models, axitinib has been reported to trigger cellular senescence both in tumor as well as in normal cells, through a mechanism involving intracellular reactive oxygen species (ROS) accumulation and activation of Ataxia Telangiectasia Mutated kinase (ATM). Limiting axitinib‐dependent ROS increase by antioxidants prevents senescence specifically in normal cells, without affecting tumor cells. Methods We used brain tumor xenografts obtained by engrafting Glioma Stem Cells (GSCs) into the brain of immunocompromised mice, to investigate the hypothesis that the antioxidant molecule N‐Acetyl‐L‐Cysteine (NAC) might be used to reduce senescence‐associated adverse effects of axitinib treatment without altering its anti‐tumor activity. Results We demonstrate that the use of the antioxidant molecule N‐Acetyl‐Cysteine (NAC) in combination with axitinib stabilizes tumor microvessels in GBM tumor orthotopic xenografts, eventually resulting in vessel normalization, and protects liver vasculature from axitinib‐dependent toxicity. Conclusion Overall, we found that NAC co‐treatment allows vessel normalization in brain tumor vessels and exerts a protective effect on liver vasculature, therefore minimizing axitinib‐dependent toxicity. Schematic representation of the experimental approach. In GBM preclinical models, we observed the efficacy of NAC cotreatment in protecting vessels from axitinib‐induced toxicity.

Modulation of endogenous neurogenesis is regarded as a promising challenge in neuroprotection. In the rat model of hippocampal neurodegeneration obtained by Trimethyltin (TMT) administration (8 mg/kg), characterised by selective pyramidal cell loss, enhanced neurogenesis, seizures and cognitive impairment, we previously demonstrated a proliferative role of exogenous neuropeptide Y (NPY), on dentate progenitors in the early phases of neurodegeneration. To investigate the functional integration of newly-born neurons, here we studied in adult rats the long-term effects of intracerebroventricular administration of NPY (2 µg/2 µl, 4 days after TMT-treatment), which plays an adjuvant role in neurodegeneration and epilepsy. Our results indicate that 30 days after NPY administration the number of new neurons was still higher in TMT+NPY-treated rats than in control+saline group. As a functional correlate of the integration of new neurons into the hippocampal network, long-term potentiation recorded in Dentate Gyrus (DG) in the absence of GABAA receptor blockade was higher in the TMT+NPY-treated group than in all other groups. Furthermore, qPCR analysis of Kruppel-like factor 9, a transcription factor essential for late-phase maturation of neurons in the DG, and of the cyclin-dependent kinase 5, critically involved in the maturation and dendrite extension of newly-born neurons, revealed a significant up-regulation of both genes in TMT+NPY-treated rats compared with all other groups. To explore the early molecular events activated by NPY administration, the Sonic Hedgehog (Shh) signalling pathway, which participates in the maintenance of the neurogenic hippocampal niche, was evaluated by qPCR 1, 3 and 5 days after NPY-treatment. An early significant up-regulation of Shh expression was detected in TMT+NPY-treated rats compared with all other groups, associated with a modulation of downstream genes. Our data indicate that the neurogenic effect of NPY administration during TMT-induced neurodegeneration involves early Shh pathway activation and results in a functional integration of newly-generated neurons into the local circuit.

Hippocampal dysfunction plays a central role in neurodevelopmental disorders, resulting in severe impairment of cognitive abilities, including memory and learning. On this basis, developmental studies represent an important tool both to understanding the cellular and molecular phenomena underlying early hippocampal damage and to study possible therapeutic interventions, that may modify the progression of neuronal death. Given the modulatory role played by 17β-estradiol (E2) on hippocampal functions and its neuroprotective properties, the present study investigates the effects of pretreatment with E2 in a model of neonatal hippocampal injury obtained by trimethyltin (TMT) administration, characterized by neuronal loss in CA1 and CA3 subfields and astroglial and microglial activation. At post-natal days (P)5 and P6 animals received E2 administration (0.2 mg/kg/die i.p.) or vehicle. At P7 they received a single dose of TMT (6.5 mg/kg i.p.) and were sacrificed 72 h (P10) or 7 days after TMT treatment (P14). Our findings indicate that pretreatment with E2 exerts a protective effect against hippocampal damage induced by TMT administration early in development, reducing the extent of neuronal death in the CA1 subfield, inducing the activation of genes involved in neuroprotection, lowering the neuroinflammatory response and restoring neuropeptide Y- and parvalbumin- expression, which is impaired in the early phases of TMT-induced damage. Our data support the efficacy of estrogen-based neuroprotective approaches to counteract early occurring hippocampal damage in the developing hippocampus.

Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

Due to its abundance, easy retrieval, and plasticity characteristics, adipose-tissue-derived stromal cells (ATSCs) present unquestionable advantages over other adult-tissue-derived stem cells. Based on the in silico analysis of our previous data reporting the ATSC-specific expression profiles, the present study attempted to clarify and validate at the functional level the expression of the neurospecific genes expressed by ATSC both in vitro and in vivo. This allowed evidencing that ATSCs express neuro-specific trophins, metabolic genes, and neuroprotective molecules. They were in fact able to induce neurite outgrowth in vitro, along with tissue-specific commitment along the neural lineage and the expression of the TRKA neurotrophin receptor in vivo. Our observation adds useful information to recent evidence proposing these cells as a suitable tool for cell-based applications in neuroregenerative medicine.

Neuroinflammation is one of the major players in amyotrophic lateral sclerosis (ALS) pathogenesis, and astrocytes are significantly involved in this process. The astrocytic protein S100B can be released in pathological states activating the receptor for advanced glycation end products (RAGE). Different indications point to an aberrant expression of S100B and RAGE in ALS. In this work, we observed that S100B and RAGE are progressively and selectively upregulated in astrocytes of diseased rats with a tissue-specific timing pattern, correlated to the level of neurodegeneration. The expression of the full-length and soluble RAGE isoforms could also be linked to the degree of tissue damage. The mere presence of mutant SOD1 is able to increase the intracellular levels and release S100B from astrocytes, suggesting the possibility that an increased astrocytic S100B expression might be an early occurring event in the disease. Finally, our findings indicate that the protein may exert a proinflammatory role in ALS, since its inhibition in astrocytes derived from SOD1G93A mice limits the expression of reactivity-linked/proinflammatory genes. Thus, our results propose the S100B-RAGE axis as an effective contributor to the pathogenesis of the disease, suggesting its blockade as a rational target for a therapeutic intervention in ALS.