Epigenetics and immune cells in medulloblastoma

: Medulloblastoma (MB) is a highly malignant childhood tumor of the cerebellum. Transcriptional and epigenetic signatures have classified MB into four molecular subgroups, further stratified into biologically different subtypes with distinct somatic copy-number aberrations, driver genes, epigenetic alterations, activated pathways, and clinical outcomes. The brain tumor microenvironment (BTME) is of importance to regulate a complex network of cells, including immune cells, involved in cancer progression in brain malignancies. MB was considered with a "cold" immunophenotype due to the low influx of immune cells across the blood brain barrier (BBB). Recently, this assumption has been reconsidered because of the identification of infiltrating immune cells showing immunosuppressive phenotypes in the BTME of MB tumors. Here, we are providing a comprehensive overview of the current status of epigenetics alterations occurring during cancer progression with a description of the genomic landscape of MB by focusing on immune cells within the BTME. We further describe how new immunotherapeutic approaches could influence concurring epigenetic mechanisms of the immunosuppressive cells in BTME. In conclusion, the modulation of these molecular genetic complexes in BTME during cancer progression might enhance the therapeutic benefit, thus firing new weapons to fight MB

MicroRNA 199b-5p delivery through stable nucleic acid lipid particles (SNALPs) in tumorigenic cell lines

MicroRNA (miR)-199b-5p has been shown to regulate Hes-1, a downstream effector of the canonical Notch and noncanonical SHH pathways, whereby it impairs medulloblastoma (MB) cancer stem cells (CSCs) through a decrease in the CD133+/CD15+ cell population. Here, we have developed stable nucleic acid lipid particles (SNALPs) that encapsulate miR-199b-5p. The efficacy of the miR- 199b-5p delivery by these SNALPs is demonstrated by significant impairment of Hes-1 levels and CSC markers in a range of different tumorigenic cell lines: colon (HT- 29, CaCo-2, and SW480), breast (MDA-MB231T and MCF-7), prostate (PC-3), glioblastoma (U-87), and MB(Daoy, ONS-76, and UW-228). After treatment with SNALP miR-199b-5p, there is also impairment of cell pro- liferation and no signs of apoptosis, as measured by cas- pases 3/7 activity and annexin V fluorescence cell sorter analyses. These data strengthen the importance of such carriers for miRNA delivery, which show no cytotoxic effects and provide optimal uptake into cells. Thus, efficient target downregulation in different tumorigenic cell lines will be the basis for future preclinical studies

RIP1-HAT1-SirT complex identification and targeting in treatment and prevention of cancer

Purpose: Alteration in cell death is a hallmark of cancer. A functional role regulating survival, apoptosis, and necroptosis has been attributed to RIP1/3 complexes.Experimental Design: We have investigated the role of RIP1 and the effects of MC2494 in cell death induction, using different methods as flow cytometry, transcriptome analysis, immunoprecipitation, enzymatic assays, transfections, mutagenesis, and in vivo studies with different mice models.Results: Here, we show that RIP1 is highly expressed in cancer, and we define a novel RIP1/3-SIRT1/2-HAT1/4 complex. Mass spectrometry identified five acetylations in the kinase and death domain of RIP1. The novel characterized pan-SIRT inhibitor, MC2494, increases RIP1 acetylation at two additional sites in the death domain. Mutagenesis of the acetylated lysine decreases RIP1-dependent cell death, suggesting a role for acetylation of the RIP1 complex in cell death modulation. Accordingly, MC2494 displays tumor-selective potential in vitro, in leukemic blasts ex vivo, and in vivo in both xenograft and allograft cancer models. Mechanistically, MC2494 induces bona fide tumor-restricted acetylated RIP1/caspase-8-mediated apoptosis. Excitingly, MC2494 displays tumor-preventive activity by blocking 7,12-dimethylbenz(α)anthracene-induced mammary gland hyperproliferation in vivoConclusions: These preventive features might prove useful in patients who may benefit from a recurrence-preventive approach with low toxicity during follow-up phases and in cases of established cancer predisposition. Thus, targeting the newly identified RIP1 complex may represent an attractive novel paradigm in cancer treatment and prevention

Early targets of miR-34a in neuroblastoma

Several genes encoding for proteins involved in proliferation, invasion, and apoptosis are known to be direct miR-34a targets. Here, we used proteomics to screen for targets of miR-34a in neuroblastoma (NBL), a childhood cancer that originates from precursor cells of the sympathetic nervous system. We examined the effect of miR-34a overexpression using a tetracycline inducible system in two NBL cell lines (SHEP and SH-SY5Y) at early time points of expression (6, 12, and 24 h). Proteome analysis using post-metabolic labeling led to the identification of 2,082 proteins, and among these 186 were regulated (112 proteins down-regulated and 74 up-regulated). Prediction of miR-34a targets via bioinformatics showed that 32 transcripts held miR-34a seed sequences in their 3′-UTR. By combining the proteomics data with Kaplan Meier geneexpression studies, we identified seven new gene products (ALG13, TIMM13, TGM2, ABCF2, CTCF, Ki67, and LYAR) that were correlated with worse clinical outcomes. These were further validated in vitro by 3′-UTR seed sequence regulation. In addition, Michigan Molecular Interactions searches indicated that together these proteins affect signaling pathways that regulate cell cycle and proliferation, focal adhesions, and other cellular properties that overall enhance tumor progression (including signaling pathways such as TGF-β, WNT, MAPK, and FAK). In conclusion, proteome analysis has here identified early targets of miR-34a with relevance to NBL tumorigenesis. Along with the results of previous studies, our data strongly suggest miR-34a as a useful tool for improving the chance of therapeutic success with NBL

H-Prune through GSK-3β interaction sustains canonical WNT/β-catenin signaling enhancing cancer progression in NSCLC.

H-Prune hydrolyzes short-chain polyphosphates (PPase activity) together with an hitherto cAMP-phosphodiesterase (PDE), the latest influencing different human cancers by its overexpression. H-Prune promotes cell migration in cooperation with glycogen synthase kinase-3 (Gsk-3β). Gsk-3β is a negative regulator of canonical WNT/β-catenin signaling. Here, we investigate the role of Gsk-3β/h-Prune complex in the regulation of WNT/β-catenin signaling, demonstrating the h-Prune capability to activate WNT signaling also in a paracrine manner, through Wnt3a secretion. In vivo study demonstrates that h-Prune silencing inhibits lung metastasis formation, increasing mouse survival. We assessed h-Prune levels in peripheral blood of lung cancer patients using ELISA assay, showing that h-Prune is an early diagnostic marker for lung cancer. Our study dissects out the mechanism of action of h-Prune in tumorigenic cells and also sheds light on the identification of a new therapeutic target in non-small-cell lung cancer

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-β-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3

Entamoeba lysyl-tRNA Synthetase Contains a Cytokine-Like Domain with Chemokine Activity towards Human Endothelial Cells

Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity

Molecular analysis of pediatric brain tumors identifies microRNAs in pilocytic astrocytomas that target the MAPK and NF-kappa B pathways

RT-qPCR confirms (a) up-regulation of miR-34a, miR-146a, miR-542-3p and miR-503 in pilocytic astrocytomas. (b) low expression of miR-124*, miR-129 and miR-129* in pilocytic astrocytomas. Relative expression shown as Log2 fold change compared to normal adult cerebellum and frontal lobe (normalized to miR-423-3p). Data represent two technical replicates ± SD. (ZIP 516 kb

Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish

Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies

Recapitulation of tumor heterogeneity and molecular signatures in a 3D brain cancer model with decreased sensitivity to histone deacetylase inhibition

INTRODUCTION Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS). METHODS CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat. RESULTS Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches. CONCLUSIONS Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system