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Purpose: Somatic mutations in the human cytosolic isocitrate dehydrogenase 1 (IDH1) gene cause profound changes in cell metabolism and are a common feature of gliomas with unprecedented predictive and prognostic impact. Fourier-transform infrared (FT-IR) spectroscopy addresses the molecular composition of cells and tissue and was investigated to deduct the IDH1 mutation status. Experimental Design: We tested the technique on human cell lines that were transduced with wild-type IDH1 or mutated IDH1 and on 34 human glioma samples. IR spectra were acquired at 256 positions from cell pellets or tissue cryosections. Moreover, IR spectra were obtained from fresh, unprocessed biopsies of 64 patients with glioma. Results: IDH1 mutation was linked to changes in spectral bands assigned to molecular groups of lipids and proteins in cell lines and human glioma. The spectra of cryosections of brain tumor samples showed high interpatient variability, for example, bands related to calcifications at 1113 cm−1. However, supervised classification recognized relevant spectral regions at 1103, 1362, 1441, 1485, and 1553 cm−1 and assigned 88% of the tumor samples to the correct group. Similar spectral positions allowed the classification of spectra of fresh biopsies with an accuracy of 86%. Conclusions: Here, we show that vibrational spectroscopy reveals the IDH1 genotype of glioma. Because it can provide information in seconds, an implementation into the intraoperative workflow might allow simple and rapid online diagnosis of the IDH1 genotype. The intraoperative confirmation of IDH1 mutation status might guide the decision to pursue definitive neurosurgical resection and guide future in situ therapies of infiltrative gliomas. Clin Cancer Res; 24(11); 2530–8. ©2017 AACR. See related commentary by Hollon and Orringer, p. 2467

Background Survivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors. It has been hypothesized that Survivin facilitates carcinogenesis by inhibition of apoptosis resulting in improved survival of tumorigenic progeny. Additionally, Survivin plays an essential role during mitosis. Together with its molecular partners Aurora B, Borealin and inner centromere protein it secures bipolar chromosome segregation. However, whether increased Survivin levels contribute to progression of tumors by inducing chromosomal instability remains unclear. Methods We overexpressed Survivin in U251-MG, SVGp12, U87-MG, HCT116 and p53-deficient U87-MGshp53 and HCT116p53−/− cells. The resulting phenotype was investigated by FACS-assisted cell cycle analysis, Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and in a U251-MG xenograft model using immune-deficient mice. Results Overexpression of Survivin affected cells with knockdown of p53, cells harboring mutant p53 and SV40 large T antigen, respectively, resulting in the increase of cell fractions harboring 4n and >4n DNA contents. Increased γH2AX levels, indicative of DNA damage were monitored in all Survivin-transduced cell lines, but only in p53 wild type cells this was accompanied by an attenuated S-phase entry and activation of p21waf/cip. Overexpression of Survivin caused a DNA damage response characterized by increased appearance pDNA-PKcs foci in cell nuclei and elevated levels of pATM S1981 and pCHK2 T68. Additionally, evolving structural chromosomal aberrations in U251-MG cells transduced with Survivin indicated a DNA-repair by non-homologous end joining recombination. Subcutaneous transplantation of U251-MG cells overexpressing Survivin and mycN instead of mycN oncogene alone generated tumors with shortened latency and decreased apoptosis. Subsequent SKY-analysis of Survivin/mycN-tumors revealed an increase in structural chromosomal aberrations in cells when compared to mycN-tumors. Conclusions Our data suggest that increased Survivin levels promote adaptive evolution of tumors through combining induction of genetic heterogeneity with inhibition of apoptosis. Electronic supplementary material The online version of this article (10.1186/s12885-017-3932-y) contains supplementary material, which is available to authorized users.

The equilibrium between proliferation and apoptosis is tightly balanced to maintain tissue homeostasis in normal tissues and even in tumors. Achieving and maintaining such a balance is important for cancer regrowth and spreading after cytotoxic treatments. Caspase-3 activation and tumor cell death following anticancer therapy as well as accompanying cell death pathways are well characterized, but their association to homeostasis of cancerous tissue and tumor progression remains poorly understood. Here we proposed a novel mechanism of cancer spreading induced by caspase-3. RhoGDIβ, known as a direct cleavage substrate of caspase-3, is overexpressed in many epithelial cancers. The N-terminal-truncated RhoGDIβ (ΔN-RhoGDIβ) is accumulated in caspase-3-activated cells. Stable expression of ΔN-RhoGDIβ in HeLa cells did not induce apoptosis, but impaired directional cell migration in a wound-healing assay accompanied by a perturbed direction of cell division at the wound edge. Subcellular protein fractionation experiments revealed that ΔN-RhoGDIβ but not wild-type RhoGDIβ was present in the detergent-soluble cytoplasmic and nuclear fractions and preferentially associated with Cdc42. Furthermore, Cdc42 activity was constitutively inhibited by stable expression of ΔN-RhoGDIβ, resulting in increased radiation-induced compensatory proliferation linking to RhoA activation. Thus, ΔN-RhoGDIβ dominant-negatively regulates Cdc42 activity and contributes to loss of polarity-related functions. The caspase-3-cleaved RhoGDIβ is a possible determinant to promote cancer spreading due to deregulation of directional organization of tumor cell population and inhibition of default equilibrium between proliferation and apoptosis after cytotoxic damage. J. Cell. Physiol. 231: 2493-2505, 2016. © 2016 Wiley Periodicals, Inc.

NK cells are emerging as new effectors for immunotherapy of cancer. In particular, the genetic engraftment of chimeric Ag receptors (CARs) in NK cells is a promising strategy to redirect NK cells to otherwise NK cell–resistant tumor cells. On the basis of DNAX-activation protein 12 (DAP12), a signaling adaptor molecule involved in signal transduction of activating NK cell receptors, we generated a new type of CAR targeting the prostate stem cell Ag (PSCA). We demonstrate in this article that this CAR, designated anti–PSCA-DAP12, consisting of DAP12 fused to the anti-PSCA single-chain Ab fragment scFv(AM1) confers improved cytotoxicity to the NK cell line YTS against PSCA-positive tumor cells when compared with a CAR containing the CD3ζ signaling chain. Further analyses revealed phosphorylation of the DAP12-associated ZAP-70 kinase and IFN-γ release of CAR-engineered cells after contact with PSCA-positive target cells. YTS cells modified with DAP12 alone or with a CAR bearing a phosphorylation-defective ITAM were not activated. Notably, infused YTS cells armed with anti–PSCA-DAP12 caused delayed tumor xenograft growth and resulted in complete tumor eradication in a significant fraction of treated mice. The feasibility of the DAP12-based CAR was further tested in human primary NK cells and confers specific cytotoxicity against KIR/HLA-matched PSCA-positive tumor cells, which was further enhanced by KIR-HLA mismatches. We conclude that NK cells engineered with DAP12-based CARs are a promising tool for adoptive tumor immunotherapy.

The mitotic Aurora B kinase is overexpressed in tumors and various inhibitors for Aurora B are currently under clinical assessments. However, when considering Aurora B kinase inhibitors as anticancer drugs, their mode of action and the role of p53 status as a possible predictive factor for response still needs to be investigated. In this study, we analyzed the effects of selective Aurora B inhibition using AZD1152-HQPA/Barasertib (AZD1152) on HCT116 cells, U87-MG, corresponding isogenic p53-deficient cells and a primary glioblastoma cell line. AZD1152 treatment caused polyploidy and non-apoptotic cell death in all cell lines irrespective of p53 status and was accompanied by poly-merotelic kinetochore-microtubule attachments and DNA damage. In p53 wild-type cells a DNA damage response induced an inefficient pseudo-G1 cell cycle arrest, which was not able to halt ongoing endoreplication of cells. Of note, release of tumor cells from AZD1152 resulted in recovery of aneuploid progenies bearing numerical and structural chromosomal aberrations. Yet, AZD1152 treatment enhanced death receptor TRAIL-R2 levels in all tumor cell lines investigated. A concomitant increase of the activating natural killer (NK) cell ligand MIC A/B in p53-deficient cells and an induction of FAS/CD95 in cells containing p53 rendered AZD1152-treated cells more susceptible for NK-cell-mediated lysis. Our study mechanistically explains a p53-independent mode of action of a chemical Aurora B inhibitor and suggests a potential triggering of antitumoral immune responses, following polyploidization of tumor cells, which might constrain recovery of aneuploid tumor cells.

BACKGROUND: The AKT1 mutation was newly described in a subset of meningiomas and inhibitors of this mutation have shown promise in clinical trials in multiple cancer types. We sought to determine the frequency of the AKT1 mutation as well as the expression level of multiple growth factor receptors in a large series of patients with Anaplastic meningiomas (AM) WHO °III. METHODS: Patients with AM °III were tested for the AKT1 (E17K) mutation using PCR technique. Additionally, the expression level of the epidermal growth factor receptor (EGFR), the platelet derived growth factor receptor 1alpha (PDGFR) and the vascular endothelial growth factor receptor (VEGFR) was detected by immunohistochemistry (IHC) using the indirect peroxidase technique on tissue multi arrays (TMA) of paraffin-embedded specimens. Staining was evaluated using a semi-quantitative scoring system. Chi-square test was used for statistical evaluation. RESULTS: We identified 22 AM °III patients with 45 tumors (median follow-up of 8 years). None of the examined 45 tumor samples in this cohort showed an AKT1 mutation (0%). Regarding the IHC, recurrent AM showed increasing proliferation with each recurrence. Overexpression of EGFR was associated with malignancy (p < 0.01) and increased with recurrence (median score 2.3). The median overall survival rate for patients with EGFR overexpression was 2.2 years. In contrast, the overall survival of patients with low EGFR expression was not reached yet (p < 0.05). While PDGFR and VEGFR scores were high in malignant tumors, the difference in comparison with benign meningiomas was not significant. A further finding is a marked survival benefit in our patients cohort in respect to the extent of resection when repeated operations were performed (p = 0.025). CONCLUSIONS: Somatic AKT1 mutations are absent in anaplastic meningiomas WHO grade III. Additionally, our data demonstrate that the overexpression of EGFR in AM might make this receptor valuable as a therapeutic target for their treatment.

Malignant glioma represents the most common primary adult brain tumor in Western industrialized countries. Despite aggressive treatment modalities, the median survival duration for patients with glioblastoma multiforme (GBM), the highest grade malignant glioma, has not improved significantly over past decades. One promising approach to deal with GBM is the inactivation of proteins essential for survival or progression of glioma cells by means of RNA interference (RNAi) techniques. A likely candidate for an RNAi therapy of gliomas is the inhibitor of apoptosis protein survivin. Survivin is involved in 2 main cellular processes-cell division and inhibition of apoptosis. We show here that stable RNAi of survivin induced polyploidy, apoptosis, and impaired proliferation of human U343-MG, U373-MG, H4, and U87-MG cells and of primary glioblastoma cells. Proteome profiler arrays using U373-MG cells identified a novel set of differentially expressed genes upon RNAi-mediated survivin knockdown. In particular, the death receptor TRAIL R2/DR5 was strongly upregulated in survivin-depleted glioma cells, inducing an enhanced cytotoxic response of allogeneic human NK cells. Moreover, an experimental in vivo therapy using polyethylenimine (PEI)/siRNA complexes for survivin knockdown efficiently blocked tumor growth of established subcutaneous U373-MG tumors and enhanced survival of NMRI(nu/nu) mice orthopically transplanted with U87-MG cells. We conclude that survivin is functionally relevant in gliomas and that PEI-mediated exogenous delivery of siRNA targeting survivin is a promising strategy for glioblastoma therapy.

Giant cell glioblastoma (gcGB), a subtype of GB, is characterized by the presence of numerous multinucleated giant cells. The prognosis for gcGB is poor, but it may have a better clinical outcome compared with classic GB. The molecular alterations that lead to the multinucleated cell phenotype of gcGB have not been elucidated. Giant cell GB has a higher frequency of the tumor suppressor protein p53 mutations than GB, however, and a role for the mitotic Aurora B kinase has been suggested. We analyzed Aurora B expression in gcGB (n = 28) and GB (n = 54) patient tumor samples by immunohistochemistry; 17 gcGB and 22 GB samples were analyzed at the DNA and mRNA levels. No mutations in the Aurora B gene (AURKB) were found, but its mRNA and protein levels were significantly higher in gcGB than in GB. Fifty-nine percent of gcGB samples but only 18% of the GB samples showed p53 mutations. Ectopic overexpression of Aurora B induced a significant increase inthe proportion of multinucleated cells in p53 mutant U373-MG, but not in p53 wild-type U87-MG, glioma cells. RNAi of p53 in U87-MG cells led to an increase in the fraction of multinucleated cells that was further augmented by ectopic overexpression of Aurora B. These results suggest that loss of p53 function and dysregulated Aurora B protein levels might represent factors that drive the development of multinucleated cells in gcGB.

Glioblastoma multiforme (GBM) is the most prevalent malignant primary brain tumor in adults. GBM is classified as primary if it is assumed to have arisen de novo or as secondary if it progressed from lower grade astrocytoma. Previous studies have found that primary and secondary GBMs have distinct molecular and mutational profiles. Both have a grim prognosis with survival times of about a year with therapy. Although much progress in delineating the temporal order of mutations and copy number aberrations in the progression of lower grade gliomas was made in the past years, none of the studies have actively followed individual tumors through their progression. Whereas this method can detect aberrations that are prevalent in gliomas, it can miss events that are important in a subset of gliomas or are necessary for progression. The GlioMath-DD consortium is an interdisciplinary collaboration of several groups at the Technische Universität Dresden (TU-Dresden) aiming to study the progression of gliomas and to come up with a mathematical model for gliomagenesis. The work involves analyzing pairs of gliomas obtained from patients who had presented with a low grade glioma and who later had a recurrence of a higher grade glioma. All tumors were checked by pathologists and then high quality DNA and RNA material extracted and used for analysis. The tumors are analyzed for copy number variations (CNV) by array comparative genome hybridization (aCGH), while gene expression changes and small mutations are analyzed using high-throughput sequencing (RNA- and Exome-seq). The data gleaned from these experiments and from in vitro models of cell growth and spheroid formation will be used by bioinformaticians and mathematicians to infer key signaling networks and formulate a mathematical model of glioma progression. The ultimate aim of this work that spans two and a half years is to create a comprehensive model of glioma promotion and progression. Furthermore, it will pinpoint driver mutations and aberrations that contribute to this progression and eventually isolate biomarkers for diagnosis and therapy. During the conference, we will present preliminary genetic data of our ongoing study. Acknowledgements: The GlioMath-DD project (coordinator: Andreas Deutsch; SAB-Number 100098214) is funded by the European Social Fund (ESF) and the Free State of Saxony Citation Format: Evelin Schröck, Khalil Abou-El-Ardat, Ralf Wiedemuth, Michael Seifert, Alvaro Köhn-Luque, Mirjam Ingargiola, Kristin Stirnnagel, Alexander Krüger, Wolfgang Nagel, Kathrin Geiger, Andreas Beyer, Leoni A. Kunz-Schughart, Gabriele Schackert, Achim Temme, Barbara Klink, Andreas Deutsch. GlioMath-DD: A multidisciplinary approach to study glioma evolution and identify targets for individualized therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5341. doi:10.1158/1538-7445.AM2014-5341

INTRODUCTION: Diffuse gliomas are the most prevalent brain tumors in adults and cannot be cured. Amino-acid substitution at position 132 of cytoplasmic enzyme isocitrate dehydrogenase 1 (IDH1) are characteristic for lower grade gliomas and therefrom derived secondary glioblastomas (GBM). The most frequent mutation observed in low grade gliomas is IDH1 c.395G>A (p.R132H). The wild type IDH1 converts isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP). In contrast, IDH1 R132H produces high amounts of 2-hydroxyglutarate (2HG), which leads to a hypermethylation phenotype and is therefore considered to be an oncometabolite. Nevertheless, the accumulation of 2HG cannot explain all the observed phenomena in gliomas. We aimed to investigate other mechanisms by which IDH1 R132H mutation contributes to gliomagenesis. MATERIALS AND METHODS: We have stably transduced three cell lines (a established GBM cell line, a primary patient-derived GBM cell line, and an immortalized astrocyte cell line) with IDH1 c.395G>A cDNA using viral vectors. Transduction success was validated on DNA, RNA and protein levels using Sanger sequencing, allele-specific PCR and Western blots. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to measure Krebs cycle metabolite concentrations. In order to find effects independent of 2HG, we added the oncometabolite to empty vector controls. NADP+/NADPH quantification kits were used to determine the concentrations of this coenzyme. RESULTS: We confirmed the presence of IDH1 c.395G>A;p.R132H mutation in the cells transduced with the mutant variant of IDH1. There was an up to 1700-fold increase of the oncometabolite 2HG in the cells with the mutation compared to an empty vector control. The levels of glutamate significantly decreased in cells with the IDH1 mutation as well as in empty vector controls after 2HG treatment. We found a ∼50% decrease in NADPH concentration in GBM cells with the IDH1 mutation but not in astrocytes. Interestingly, there was a decrease in proliferation of GBM cells with IDH1 R132H. This effect was confirmed in 3D spheroid cell cultures. CONCLUSION: The mechanisms of how IDH1 R132H mutations contribute to gliomagenesis are still far from clear. We found that mutations in IDH1 lead to changes in Krebs cycle metabolite concentrations and energy homeostasis while decreasing proliferation in vitro. We are currently analyzing the effect of IDH1 R132H on the transcriptome level in comparison with IDH1 wt cells treated with 2HG. Based on these profiles we aim to investigate possible genetic mechanisms which are independet of the oncometabolite 2HG. These results will also be presented.