Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.Electronic supplementary materialThe online version of this article (10.1007/s00401-019-02062-4) contains supplementary material, which is available to authorized users.

Osteoprotegerin (OPG) is a recently cloned member of the tumour necrosis factor receptor family. It has been suggested that this secreted glycoprotein acts as an inhibitor of osteoclastic differentiation. Expression of OPG has previously been demonstrated in a number of tissues. However, it is still unclear whether or not OPG is expressed by human osteoblasts. We have used the RNase protection assay to demonstrate the OPG transcript in primary cultured human osteoblast-like cells, human marrow stroma cells and osteosarcoma cell lines. Furthermore, we have studied the effect of glucocorticoids on OPG mRNA levels in these cells. We demonstrate that glucocorticoids decrease the OPG transcript in a dose-and time-dependent manner. The time-course study reveals that hydrocortisone (10 6 M) decreases OPG mRNA levels within 2 h. This decrease is transient, reaching control levels again after 24 h.Our findings demonstrate that human osteoblasts express the mRNA corresponding to OPG, an inhibitor of osteoclast differentiation. The finding that OPG mRNA levels are decreased by glucocorticoids indicates that a reduced production of OPG from osteoblasts and/or marrow stroma cells could, in part, explain glucocorticoidinduced bone resorption.

Tau phosphorylation was examined in argyrophilic grain disease (AGD) by using the phosphospecific tau antibodies Thr181, Ser202, Ser214, Ser 396 and Ser422, and antibodies to non-phosphorylated and phosphorylated mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK), stress-activated kinase (SAPK), c-Jun Nterminal kinase (JNK), p38 kinase (p-38), ␣-calcium/calmodulin-dependent kinase II (␣CaM kinase II), and glycogen synthase kinase-3 (GSK-3), all of which regulate phosphorylation at specific sites of tau. This is the first study in which the role of protein kinases in tau phosphorylation has been examined in AGD.Hyperphosphorylated tau accumulated in grains and pre-tangles in the hippocampus, dentate gyrus, entorhinal and trans-entorhinal cortices, and amygdala in all cases. Ballooned neurons in the amygdala, entorhinal, insular and cingulate cortex, and claustrum contained ␣B-crystallyn and phosphorylated neurofilament epitopes. Some astrocytes and scattered oligodendrocytes containing coiled bodies were recognized with anti-tau antibodies. A few tangles were observed in the entorhinal cortex and hippocampus corresponding to Alzheimer's disease (AD) stages I-III of Braak and Braak. None of the present cases was associated with progressive supranuclear palsy or with ␣-synuclein pathology. Two bands of phospho-tau of 64 and 68 kDa were observed in Western blots of sarkosyl-insoluble fractions enriched with abnormal filaments in AGD, a pattern that contrasts with the 4-band pattern obtained in AD.No modifications in the expression of non-phosphorylated MEK-1, ERK2 and GSK-3␣/␤, as revealed by immunohistochemistry, were seen in AGD, but sarkosyl-insoluble fractions were particularly enriched in JNK-1 and ␣CaM kinase II. Increased expression of the phosphorylated (P) forms of MAPK/ERK, SAPK/JNK, p38 and GSK-3␤ was found in grains and tau-containing cells in AGD. MAPK/ERK-P immunoreactivity was observed in pre-tangles and, diffusely, in the cytoplasm of ballooned neurons, but not in grains. Strong SAPK/JNK-P and P38-P, and moderate GSK-3b-P immunoreactivities selectively occured in grains, in neurons with pre-tangles and in the peripheral region of the cytoplasm of ballooned neurons. MAPK/ERK-P, SAPK/JNK-P, p38-P and GSK-3␤-P were expressed in tau-containing astrocytes and in oligodendrocytes with coiled bodies. Western blots revealed kinase expression in sarkosyl-insoluble fractions but none of the phospho-kinase antibodies recognized hyper-phosphorylated tau protein.These findings indicate complex, specific profiles of tau phosphorylation and concomitant activation of precise kinases that have the capacity to phosphorylate tau at specific sites in AGD. These kinases co-localize abnormal tau in selected structures and cells, including neurons with pre-tangles, ballooned neurons, astrocytes and oligodendrocytes.Most of these kinases are involved in cell death and cell survival in certain experimental paradigms. However, double-labeling studies with the method of in situ end-labeling of...

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BackgroundVCN-01 is an oncolytic adenovirus (Ad5 based) designed to replicate in cancer cells with dysfunctional RB1 pathway, express hyaluronidase to enhance virus intratumoral spread and facilitate chemotherapy and immune cells extravasation into the tumor. This phase I clinical trial was aimed to find the maximum tolerated dose/recommended phase II dose (RP2D) and dose-limiting toxicity (DLT) of the intravenous delivery of the replication-competent VCN-01 adenovirus in patients with advanced cancer.MethodsPart I: patients with advanced refractory solid tumors received one single dose of VCN-01. Parts II and III: patients with pancreatic adenocarcinoma received VCN-01 (only in cycle 1) and nab-paclitaxel plus gemcitabine (VCN-concurrent on day 1 in Part II, and 7 days before chemotherapy in Part III). Patients were required to have anti-Ad5 neutralizing antibody (NAbs) titers lower than 1/350 dilution. Pharmacokinetic and pharmacodynamic analyses were performed.Results26% of the patients initially screened were excluded based on high NAbs levels. Sixteen and 12 patients were enrolled in Part I and II, respectively: RP2D were 1×1013 viral particles (vp)/patient (Part I), and 3.3×1012 vp/patient (Part II). Fourteen patients were included in Part III: there were no DLTs and the RP2D was 1×1013 vp/patient. Observed DLTs were grade 4 aspartate aminotransferase increase in one patient (Part I, 1×1013 vp), grade 4 febrile neutropenia in one patient and grade 5 thrombocytopenia plus enterocolitis in another patient (Part II, 1×1013 vp). In patients with pancreatic adenocarcinoma overall response rate were 50% (Part II) and 50% (Part III). VCN-01 viral genomes were detected in tumor tissue in five out of six biopsies (day 8). A second viral plasmatic peak and increased hyaluronidase serum levels suggested replication after intravenous injection in all patients. Increased levels of immune biomarkers (interferon-γ, soluble lymphocyte activation gene-3, interleukin (IL)-6, IL-10) were found after VCN-01 administration.ConclusionsTreatment with VCN-01 is feasible and has an acceptable safety. Encouraging biological and clinical activity was observed when administered in combination with nab-paclitaxel plus gemcitabine to patients with pancreatic adenocarcinoma.Trial registration numberNCT02045602.

The transcription factor C/EBP alpha, a member of the CCAAT/enhancer-binding protein family, is highly expressed in the liver and in adipose tissue. The aim of this study was to determine if C/EBP alpha is expressed in rat growth cartilage. The expression pattern of C/EBP alpha in monolayer-cultured growth plate chondrocytes was similar to that of C/EBP alpha during hepatocyte and preadipocyte differentiation. Immunohistochemistry with a polyclonal antibody for C/EBP alpha revealed that the C/EBP alpha protein is present in the perichondrial ring, in the germinal layer of the growth plate and on the surface of the articular cartilage. The growth hormone (GH) receptor has a similar distribution in the rat tibial growth plate, and hypophysectomised rats were used to investigate a possible connection between C/EBP alpha and GH. C/EBP alpha mRNA levels were decreased in rib cartilage after hypophysectomy. However, GH treatment did not counteract this effect, indicating that other pituitary hormones regulate the C/EBP alpha mRNA levels in growth plate cartilage. We thus demonstrate, for the first time, that C/EBP alpha is expressed in cartilage. The finding that C/EBP alpha, like the GH receptor, is predominantly expressed in stem cell areas of the rat growth plate indicates a possible functional role for C/EBP alpha during early chondrogenic differentiation.

Expression of IGFBP2 (Insulin-like Growth Factor Binding Protein 2) has been positively correlated with glioma progression. Although the proteolysis of IGFBP2 has been widely recognized, with consequences as a major modulator of IGFII signaling, the relevance of this post-translational modification has not been well studied in tumors. Using an in vivo proteomic approach by Isotope-Coded Protein Label (ICPL), we identified IGFBP2 as a target of the extracellular protease ADAMTS1 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 1). Notably, the proteolytic pattern of IGFBP2 was also detected in human glioma culture cells and, more importantly, in all glioma samples evaluated. In addition, high expression of ADAMTS1 correlates with higher levels of cleaved IGFBP2 in glioblastoma multiforme cases. Using gene expression public databases, we confirmed that IGFBP2 is a poor prognosis marker for gliomas, and we also observed an important contribution of ADAMTS1.Finally, we showed the impact of ADAMTS1 on IGFII-mediated IGF1R phosphorylation and cellular migration. Our results support a functional interaction between IGFBP2 and ADAMTS1 and suggest the need to evaluate post-translational modifications of IGFBP2 in glioma, in order to approach new therapies.

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