Il controllo epigenetico di geni oncosoppressori nelle cellule staminali neuronali normali e di medulloblastoma

Le cellule staminali cerebellari normali fanno parte della grande famiglia delle cellule staminali somatiche, deputate a sostituire le cellule che muoiono per omeostasi fisiologica del tessuto o per un danno. Tali cellule mantengono alcune delle caratteristiche versatili delle cellule staminali embrionali. In particolare sono dotate della capacità di auto-rinnovamento indefinito e di multipotenza, difatti sono in grado di differenziare in più linee cellulari. Le cellule staminali neuronali sono state identificate in molte aree del cervello adulto e risiedono anche nel cervelletto postnatale. Meccanismi aberranti di sviluppo neuronale e cerebellare possono portare alla formazione neoplasie che nel cervelletto prendono il nome di Medulloblastoma, il più frequente tumore cerebrale maligno in età infantile. In tale neoplasia si può riscontrare un’attivazione aberrante della via del segnale di Hedgehog come conseguenza di cambiamenti genetici ed epigenetici che colpiscono vari componenti della via del segnale. Negli ultimi anni è stato proposto un modello alternativo per l’evoluzione del cancro. Questo nuovo modello stabilisce l’esistenza di un ordine gerarchico dove una cellula tessuto-specifica, chiamata “cancer stem cell”, acquisisce o mantiene le proprietà di auto-rinnovamento, multipotenza e di generazione di un tumore sia in vitro che in vivo. Il miR-326 è un noto inibitore della via del segnale di Hedgehog nei granuli cerebellari (Ferretti et al. 2008), ne abbiamo valutato l’espressione nelle cellule staminali neuronali normali e di medulloblastoma sia umane che murine e, come atteso, il miR-326 è poco espresso in tutti i modelli cellulari analizzati. Inoltre, tali cellule, se sottoposte a stimoli differenziativi, esprimono nuovamente il miR-326 evidenziandone un’ipotetica funzione nel differenziamento cerebellare. Abbiamo confermato l'azione di tale microRNA come inibitore della via del segnale di Hegehog non solo mediante l'inibizione di Smoothened ma anche mediante il nuovo target da noi identificato, Gli2. Abbiamo inoltre riscontrato una netta riduzione della proliferazione e della capacità clonogenica delle cellule staminali in presenza del miR-326. Il miR-326 è localizzato all’interno del primo introne del gene della β-arrestina1. Valutando l’espressione della β-arrestina1 nei vari contesti cellulari analizzati, abbiamo riscontrato le stesse modulazioni del miR-326 confermando l’ipotesi bioinformatica che i due geni fossero sotto il controllo dello stesso promotore. Abbiamo confermato che la β-arrestina1 agisce come inibitore del ciclo cellulare attivando p27, come già descritto in letteratura. Abbiamo caratterizzando la regione regolatoria comune dei geni miR-326 e β-arrestina1 evidenziando come tali geni siano regolati mediante metilazione del DNA, difatti la loro espressione viene ripristinata grazie all’utilizzo dell’agente demetilante 5’- aza-2’deossicitidina. Essendo noto in letteratura la correlazione di EZH2 con la metilazione del DNA e la sua funzione nelle modificazioni istoniche, abbiamo dimostrato la sua presenza sulla regione regolatoria ed anche la contemporanea presenza di un dominio bivalente ad indicare un ulteriore livello di regolazione trascrizionale ed a sottilineare l’importanza di tali geni per il differenziamento neuronale. Data la possibile implicazione nella proliferazione delle cellule staminali di medulloblastoma causata dalla deregolazione dell’espressione dei due geni, abbiamo utilizzato un nuovo composto, MC2055, inibitore di EZH2. Abbiamo dimostrato che grazie a trattamenti con tale composto, otteniamo la risoluzione del dominio bivalente e, quindi, l’espressione dei due geni con conseguente arresto della proliferazione

Consequences of simulated microgravity in neural stem cells: biological effects and metabolic response.

Objective: Microgravity was often shown to cause cell damage and impair cell cycle in a variety of biological systems. Since the effects on the neural system were poorly investigated, we aimed to gain insight into how biological processes such as cell cycle, cell damage, stemness features and metabolic status are involved in neural stem cells (NSC) when they experience simulated microgravity. We also wished to investigate whether these modulations were transient or permanent once cells were returned to normal gravity. Methods: NSC were isolated from mouse cerebella and cultured in the Rotary Cell Culture System (RCCS) to model microgravity. We analyzed cell cycle, stress and apoptotic response. We also performed a 1H NMR-based metabolomic analysis and evaluation of stemness features of NSC in simulated microgravity and once in the returned to normogravity cell culture. Results: Biological processes and metabolic status were modulated by simulated microgravity. Cells were arrested in S-phase together with enhanced apoptosis. Metabolic changes occurred in NSC after simulated microgravity. Interestingly, these modulations were transient. Indeed, stemness features and metabolic footprint returned to basal levels after few days of culture in normal conditions. Moreover NSC clonogenic ability was not impaired. Conclusions: Our data suggest that simulated microgravity impacts on NSC biological processes, including cell cycle and apoptosis. However, NSC does not suffer from permanent damage

β-arrestin1-mediated acetylation of Gli1 regulates Hedgehog/Gli signaling and modulates self-renewal of SHH medulloblastoma cancer stem cells

Background Aberrant Sonic Hedgehog/Gli (Hh/Gli) signaling pathway is a critical regulator of Sonic hedgehog medulloblastoma (SHH-MB). Cancer stem cells (CSCs), thought to be largely responsible for tumor initiation, maintenance, dissemination and relapse, have been identified in SHH-MB. Since we previously demonstrated that Hh/Gli signaling controls CSCs features in SHH-MB and that in these tumors miR-326 is down regulated, here we investigated whether there is a functional link between Hh/Gli signaling and miR-326. Methods We evaluated β-arrestin1 (Arrb1) and its intragenic miR-326 levels in CSCs derived from SHH-MB. Subsequently, we modulated the expression of Arrb1 and miR-326 in CSCs in order to gain insight into their biological role. We also analyzed the mechanism by which Arrb1 and miR-326 control Hh/Gli signaling and self-renewal, using luciferase and protein immunoprecipitation assays. Results Low levels of Arrb1 and miR-326 represent a feature of CSCs derived from SHH-MB. We observed that re-expression of Arrb1 and miR-326 inhibits Hh/Gli signaling pathway at multiple levels, which cause impaired proliferation and self-renewal, accompanied by down regulation of Nanog levels. In detail, miR-326 negatively regulates two components of the Hh/Gli pathway the receptor Smoothened (Smo) and the transcription factor Gli2, whereas Arrb1 suppresses the transcriptional activity of Gli1, by potentiating its p300-mediated acetylation. Conclusions Our results identify a new molecular mechanism involving miR-326 and Arrb1 as regulators of SHH-MB CSCs. Specifically, low levels of Arrb1 and miR-326 trigger and maintain Hh/Gli signaling and self-renewal

Preclinical toxicology and safety pharmacology of the first-in-class GADD45β/MKK7 inhibitor and clinical candidate, DTP3

Aberrant NF-κB activity drives oncogenesis and cell survival in multiple myeloma (MM) and many other cancers. However, despite an aggressive effort by the pharmaceutical industry over the past 30 years, no specific IκBα kinase (IKK)β/NF-κB inhibitor has been clinically approved, due to the multiple dose-limiting toxicities of conventional NF-κB-targeting drugs. To overcome this barrier to therapeutic NF-κB inhibition, we developed the first-in-class growth arrest and DNA-damage-inducible (GADD45)β/mitogen-activated protein kinase kinase (MKK)7 inhibitor, DTP3, which targets an essential, cancer-selective cell-survival module downstream of the NF-κB pathway. As a result, DTP3 specifically kills MM cells, ex vivo and in vivo, ablating MM xenografts in mice, with no apparent adverse effects, nor evident toxicity to healthy cells. Here, we report the results from the preclinical regulatory pharmacodynamic (PD), safety pharmacology, pharmacokinetic (PK), and toxicology programmes of DTP3, leading to the approval for clinical trials in oncology. These results demonstrate that DTP3 combines on-target-selective pharmacology, therapeutic anticancer efficacy, favourable drug-like properties, long plasma half-life and good bioavailability, with no target-organs of toxicity and no adverse effects preclusive of its clinical development in oncology, upon daily repeat-dose administration in both rodent and non-rodent species. Our study underscores the clinical potential of DTP3 as a conceptually novel candidate therapeutic selectively blocking NF-κB survival signalling in MM and potentially other NF-κB-driven cancers

GADD45β loss ablates innate immunosuppression in cancer

T cell exclusion from the tumour microenvironment (TME) is a major barrier to overcoming immune escape. Here we identify a myeloid-intrinsic mechanism governed by the NF-κB effector molecule GADD45β that restricts tumour-associated inflammation and T cell trafficking into tumours. In various models of solid cancers refractory to immunotherapies, including hepatocellular carcinoma (HCC) and ovarian adenocarcinoma, Gadd45b inhibition in myeloid cells restored activation of pro-inflammatory tumour-associated macrophages (TAM) and intratumoural immune infiltration, thereby diminishing oncogenesis. Our results provide a basis to interpret clinical evidence that elevated expression of GADD45B confers poor clinical outcomes in most human cancers. Further, they suggest a therapeutic target in GADD45β for re-programming TAM to overcome immunosuppression and T cell exclusion from the TME

Enhanced triacylglycerol catabolism by carboxylesterase 1 promotes aggressive colorectal carcinoma

The ability to adapt to low-nutrient microenvironments is essential for tumor cell survival and progression in solid cancers, such as colorectal carcinoma (CRC). Signaling by the NF-κB transcription factor pathway associates with advanced disease stages and shorter survival in patients with CRC. NF-κB has been shown to drive tumor-promoting inflammation, cancer cell survival, and intestinal epithelial cell (IEC) dedifferentiation in mouse models of CRC. However, whether NF-κB affects the metabolic adaptations that fuel aggressive disease in patients with CRC is unknown. Here, we identified carboxylesterase 1 (CES1) as an essential NF-κB–regulated lipase linking obesity-associated inflammation with fat metabolism and adaptation to energy stress in aggressive CRC. CES1 promoted CRC cell survival via cell-autonomous mechanisms that fuel fatty acid oxidation (FAO) and prevent the toxic build-up of triacylglycerols. We found that elevated CES1 expression correlated with worse outcomes in overweight patients with CRC. Accordingly, NF-κB drove CES1 expression in CRC consensus molecular subtype 4 (CMS4), which is associated with obesity, stemness, and inflammation. CES1 was also upregulated by gene amplifications of its transcriptional regulator HNF4A in CMS2 tumors, reinforcing its clinical relevance as a driver of CRC. This subtype-based distribution and unfavorable prognostic correlation distinguished CES1 from other intracellular triacylglycerol lipases and suggest CES1 could provide a route to treat aggressive CRC

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Measurement of the inclusive isolated-photon cross section in pp collisions at √s = 13 TeV using 36 fb−1 of ATLAS data

The differential cross section for isolated-photon production in pp collisions is measured at a centre-of-mass energy of 13 TeV with the ATLAS detector at the LHC using an integrated luminosity of 36.1 fb. The differential cross section is presented as a function of the photon transverse energy in different regions of photon pseudorapidity. The differential cross section as a function of the absolute value of the photon pseudorapidity is also presented in different regions of photon transverse energy. Next-to-leading-order QCD calculations from Jetphox and Sherpa as well as next-to-next-to-leading-order QCD calculations from Nnlojet are compared with the measurement, using several parameterisations of the proton parton distribution functions. The predictions provide a good description of the data within the experimental and theoretical uncertainties. [Figure not available: see fulltext.