Search

Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.

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 X-linked histone demethylase UTX has a pivotal role in cellular and developmental processes including embryogenesis, hematopoiesis and cancer. UTX removes di- and trimethyl groups on histone H3 lysine 27, thereby regulating gene expression. But there is growing evidence that UTX displays biological functions independent of its histone demethylase activity. To elucidate these novel functions, it is of great interest to define subcellular localizations of UTX. Here we show for the first time that native UTX is primarily localized in the cytoplasm whereas ectopic GFP and Flag-tagged UTX display nuclear and cytoplasmic localization. While its epigenetic function is exerted in the nucleus, its cytoplasmic localization points to a novel function.

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.

IDH1 R132H mutations are considered to play a key role in the development of low grade gliomas and therefrom derived secondary glioblastomas (GBM). Wild type IDH1 converts isocitrate to α-ketoglutarate (a-KG) while reducing NADP+. IDH1R132H has a neomorphic enzymatic function using a-KG to generate high amounts of the oncometabolite D-2-hydroxyglutarate (D-2-HG). While the effects of D-2-HG have been subject to intense research, D-2-HG independent effects of IDH1R132H on energy homeostasis and redox status are not well studied. Here we demonstrate that IDH1R132H transduction but not D-2-HG alone leads to significantly decreased Krebs cycle metabolite concentrations and proliferation in U87 and the primary GBM cell line HT7606 as well as in immortalized astrocytes SVGp12. Furthermore, IDH1R132H mutation, but not D-2-HG treatment, resulted in a significant drop in NADPH levels in tumor cells (U87 and HT7606), whereas immortalized astrocytes retained normal NADPH levels. Since NAPDH levels can be restored via the reaction of the NAD-kinase, we analyzed NAD levels and enzymes involved in NAD-synthesis in our cell lines. Indeed we found a significant drop of NAD levels and in the activity of the NAD-dependent enzyme sirtuin in IDH1R132H mutant U87 and HT7606 but not in NADPH stable SVGp12-IDH1R132H. Interestingly, there were marked differences in expression of NAD-synthesis enzymes between the different cell-lines. In particular, NAMPT-levels were much higher in U87 and HT7606 then in astrocytes (SVGp12) and significantly decreased in U87-IDH1R132H and HT7606-IDH1R132H. Importantly, we also found decreased levels of NAMPT in primary tumor tissues and patient derived glioma cell lines with IDH1 R132H compared to wild type gliomas. Altogether our results for the first time show that the IDH1 mutation directly affects energy homeostasis and redox status in a cell-type dependent manner. We hypothesize that this leads to a drop in NADPH and NAD-levels during malignant progression, resulting in a disadvantage for proliferating tumor cells. This is in line with the favorable prognosis and good response to chemo- and radiation therapy clinically observed in IDH-mutated gliomas. Our findings suggest that the impaired metabolism in IDH1-mutant tumors might be a promising target for future therapies. Citation Format: Evelin Schrock, Julia Biedermann, Khalil Abou-El-Ardat, Matthias Lehmann, Marina Conde, Mirko Peitzsch, Susan Richter, Ralf Wiedemuth, Matthias Meinhardt, William P. J. Leenders, Christel Herold-Mende, Graeme Eisenhofer, Simone P. Niclou, Leoni Kunz-Schughart, Achim Temme, Barbara Klink. Effects of the IDH1 R132H mutation on redox status and metabolism are cell type dependent but independent from D-2-hydroxyglutarate accumulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-308.

Survivin, belonging to the family of Inhibitor of Apoptosis proteins (IAP), is overexpressed in a broad variety of different tumor entities. Besides its role and function in apoptosis, Survivin forms in mitosis with its partners Borealin, inner centromere protein (INCENP) and the enzymatically active member Aurora B kinase the chromosomal passenger complex (CPC). The main function of this complex is sensing and correcting non-bipolar microtubule-kinetochore interaction, regulating chromosome segregation and cytokinesis. Some evidence points to a role of Survivin in connecting mitosis with control of cell cycle arrest. In this study, we assessed the role of Survivin in ensuring chromosomal stability and DNA integrity in HCT116, MCF-7, U87-MG and corresponding isogenic p53-deficient cells. Knock down of Survivin, using retroviral short-hairpin RNAs, caused in all tested cell lines a heavily impaired cytokinesis resulting in polyploidy and poly-merotelic kinetochore spindle assemblies, irrespective of the p53 status. Surprisingly, cells with wild type p53 showed after Survivin RNAi an activation of p53 and accumulation of p21waf/cip and Cyclin D1, indicating G1 arrest. A detailed Flow Cytometry analysis revealed a correlation between the increased p21waf/cip expression and increasing DNA content in Survivin-depleted wild type cells compared to p53-deficient cells. Conversely, BrdU incorporation in wild type cells decreased with DNA content compared to p53-deficient cells with knock down of Survivin. Nonetheless, polyploid wild type cells even with a DNA content larger 4n were still able to incorporate BrdU, indicating transient G1 arrest after Survivin knock down. Furthermore, Survivin RNAi led in all tested cell lines to an induction of DNA lesions and caused a DNA damage response. SKY-analysis revealed numerical aberrations and a significant higher frequency of structural chromosomal aberrations indicative for DNA double strand break repair, only in Survivin-depleted cells. In conclusion, our results highlight the important role of Survivin as a chromosomal passenger protein which in concert with its molecular partners Aurora B, INCENP, and Borealin, controls chromosomal segregation by regulating bipolar spindle attachment and cytokinesis and protects cells from polyploidy and chromosomal instability. Citation Format: Ralf Wiedemuth, Barbara Klink, Evelin Schroeck, Gabriele Schackert, Achim Temme. Survivin safeguards chromosome numbers and protects from aneuploidy. [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 5089. doi:10.1158/1538-7445.AM2014-5089

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.

Background Survivin, a member of the inhibitor of apoptosis (IAP) gene family, has a dual role in mitosis and in apoptosis. It is abundantly expressed in every human tumor, compared with normal tissues. During mitosis Survivin assembles with the chromosomal passenger complex and regulates chromosomal segregation. Here, we aim to explore whether interference with the mitotic function of Survivin is linked to p53-mediated G1 cell cycle arrest and affects chromosomal stability. Methods In this study, we used HCT116, SBC-2, and U87-MG and generated corresponding isogenic p53-deficient cells. Retroviral vectors were used to stably knockdown Survivin. The resulting phenotype, in particular the mechanisms of cell cycle arrest and of initiation of aneuploidy, were investigated by Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and RNAi. Results In all cell lines Survivin-RNAi did not induce instant apoptosis but caused polyplodization irrespective of p53 status. Strikingly, polyploidization after knockdown of Survivin resulted in merotelic kinetochore spindle assemblies, γH2AX-foci, and DNA damage response (DDR), which was accompanied by a transient p53-mediated G1-arrest. That p53 wild type cells specifically arrest due to DNA damage was shown by simultaneous inhibition of ATM and DNA-PK, which abolished induction of p21waf/cip. Cytogenetic analysis revealed chromosomal aberrations indicative for DNA double strand break repair by the mechanism of non-homologous end joining (NHEJ), only in Survivin-depleted cells. Conclusion Our findings suggest that Survivin plays an essential role in proper amphitelic kinetochore-spindle assembly and that constraining Survivin’s mitotic function results in polyploidy and aneuploidy which cannot be controlled by p53. Therefore, Survivin critically safeguards chromosomal stability independently from p53.