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Intrahepatic cholangiocarcinoma (iCCA) is a rare malignancy of the intrahepatic biliary tract with a very poor prognosis. Although some clinicopathological parameters can be prognostic factors for iCCA, the molecular prognostic markers and potential mechanisms of iCCA have not been well investigated. Here, we report that the Fragile X mental retardation protein (FMRP), a RNA binding protein functionally absent in patients with the Fragile X syndrome (FXS) and also involved in several types of cancers, is overexpressed in human iCCA and its expression is significantly increased in iCCA metastatic tissues. The silencing of FMRP in metastatic iCCA cell lines affects cell migration and invasion, suggesting a role of FMRP in iCCA progression. Moreover, we show evidence that FMRP is localized at the invasive front of human iCCA neoplastic nests and in pseudopodia and invadopodia protrusions of migrating and invading iCCA cancer cells. Here FMRP binds several mRNAs encoding key proteins involved in the formation and/or function of these protrusions. In particular, we find that FMRP binds to and regulates the expression of Cortactin, a critical regulator of invadopodia formation. Altogether, our findings suggest that FMRP could promote cell invasiveness modulating membrane plasticity and invadopodia formation at the leading edges of invading iCCA cells.

In fragile X syndrome (FXS) the lack of the fragile X mental retardation protein (FMRP) leads to exacerbated signaling through the metabotropic glutamate receptors 5 (mGlu5Rs). The adenosine A2A receptors (A2ARs), modulators of neuronal damage, could play a role in FXS. A synaptic colocalization and a strong permissive interaction between A2A and mGlu5 receptors in the hippocampus have been previously reported, suggesting that blocking A2ARs might normalize the mGlu5R-mediated effects of FXS. To study the cross-talk between A2A and mGlu5 receptors in the absence of FMRP, we performed extracellular electrophysiology experiments in hippocampal slices of Fmr1 KO mouse. The depression of field excitatory postsynaptic potential (fEPSPs) slope induced by the mGlu5R agonist CHPG was completely blocked by the A2AR antagonist ZM241385 and strongly potentiated by the A2AR agonist CGS21680, suggesting that the functional synergistic coupling between the two receptors could be increased in FXS. To verify if chronic A2AR blockade could reverse the FXS phenotypes, we treated the Fmr1 KO mice with istradefylline, an A2AR antagonist. We found that hippocampal DHPG-induced long-term depression (LTD), which is abnormally increased in FXS mice, was restored to the WT level. Furthermore, istradefylline corrected aberrant dendritic spine density, specific behavioral alterations, and overactive mTOR, TrkB, and STEP signaling in Fmr1 KO mice. Finally, we identified A2AR mRNA as a target of FMRP. Our results show that the pharmacological blockade of A2ARs partially restores some of the phenotypes of Fmr1 KO mice, both by reducing mGlu5R functioning and by acting on other A2AR-related downstream targets.

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.

Glioblastomas (GBMs) are highly vascularized cancers. Transdifferentiation of GBM stem-like cells (GSCs) into GSC-derived endothelial cells (GdECs) contributes to GBM neovascularization. To dissect the molecular mechanisms and the signaling pathways underlying GSC contribution to tumor vascularization, we identified a three miRNA signature able to discriminate GSCs from GdECs by regulating different signaling pathways. DUSP8 resulted as the common target of the miRNA signature identified and is negatively regulated by miR-1825. DUSP8 is emerging as a critical negative regulator MAPKs pathway and is involved in cell oxidative stress response and apoptosis, as well as, in several diseases, including cancer. In GBM patients, DUSP8 and miR-1825 expression are inversely correlated and DUSP8 down-regulation is significantly associated with higher microvascular density and poor overall survival. Exploring the impact of DUSP8 in GSC transdifferentiation, we demonstrated that DUSP8 down-regulation interferes with MAPK pathway and affects soluble factor release. In vitro DUSP8 modulation experiments showed that DUSP8 enforced expression impairs GdEC ability to form tube-like structures. Gene expression variations induced by DUSP8 modulation affect transcripts associated with EMT pathway, confirming that DUSP8 shutdown and, therefore, the activation of MAPK pathway, is mandatory to GSC transdifferentiation. In vivo experiments demonstrated that both DUSP8 enforced expression and silencing dramatically affect gliomagenesis. Dissecting the molecular mechanisms underlying the contribution of GSCs to tumor angiogenesis might represent a chance to develop new and more efficient antiangiogenic therapeutic protocols for GBM treatment. Our findings provide a strong rationale to develop therapeutic strategies based on modulation of DUSP8 for GBM treatment.

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.