TY - JOUR A1 - Vollmuth, Nadine A1 - Schlicker, Lisa A1 - Guo, Yongxia A1 - Hovhannisyan, Pargev A1 - Janaki-Raman, Sudha A1 - Kurmasheva, Naziia A1 - Schmitz, Werner A1 - Schulze, Almut A1 - Stelzner, Kathrin A1 - Rajeeve, Karthika A1 - Rudel, Thomas T1 - c-Myc plays a key role in IFN-γ-induced persistence of Chlamydia trachomatis JF - eLife N2 - Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr. Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3β axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism. KW - Chlamydia trachomatis Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301385 VL - 11 ER - TY - JOUR A1 - Schwarz, Jessica Denise A1 - Lukassen, Sören A1 - Bhandare, Pranjali A1 - Eing, Lorenz A1 - Snaebjörnsson, Marteinn Thor A1 - García, Yiliam Cruz A1 - Kisker, Jan Philipp A1 - Schulze, Almut A1 - Wolf, Elmar T1 - The glycolytic enzyme ALDOA and the exon junction complex protein RBM8A are regulators of ribosomal biogenesis JF - Frontiers in Cell and Developmental Biology N2 - Cellular growth is a fundamental process of life and must be precisely controlled in multicellular organisms. Growth is crucially controlled by the number of functional ribosomes available in cells. The production of new ribosomes depends critically on the activity of RNA polymerase (RNAP) II in addition to the activity of RNAP I and III, which produce ribosomal RNAs. Indeed, the expression of both, ribosomal proteins and proteins required for ribosome assembly (ribosomal biogenesis factors), is considered rate-limiting for ribosome synthesis. Here, we used genetic screening to identify novel transcriptional regulators of cell growth genes by fusing promoters from a ribosomal protein gene (Rpl18) and from a ribosomal biogenesis factor (Fbl) with fluorescent protein genes (RFP, GFP) as reporters. Subsequently, both reporters were stably integrated into immortalized mouse fibroblasts, which were then transduced with a genome-wide sgRNA-CRISPR knockout library. Subsequently, cells with altered reporter activity were isolated by FACS and the causative sgRNAs were identified. Interestingly, we identified two novel regulators of growth genes. Firstly, the exon junction complex protein RBM8A controls transcript levels of the intronless reporters used here. By acute depletion of RBM8A protein using the auxin degron system combined with the genome-wide analysis of nascent transcription, we showed that RBM8A is an important global regulator of ribosomal protein transcripts. Secondly, we unexpectedly observed that the glycolytic enzyme aldolase A (ALDOA) regulates the expression of ribosomal biogenesis factors. Consistent with published observations that a fraction of this protein is located in the nucleus, this may be a mechanism linking transcription of growth genes to metabolic processes and possibly to metabolite availability. KW - ribosome biogenesis KW - Ribosomal protein gene KW - genetic screen KW - genome-wide screen KW - RBM8A KW - Y14 KW - AldoA KW - aldolase A Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290875 SN - 2296-634X VL - 10 ER - TY - JOUR A1 - Peixoto, Joana A1 - Janaki-Raman, Sudha A1 - Schlicker, Lisa A1 - Schmitz, Werner A1 - Walz, Susanne A1 - Winkelkotte, Alina M. A1 - Herold-Mende, Christel A1 - Soares, Paula A1 - Schulze, Almut A1 - Lima, Jorge T1 - Integrated metabolomics and transcriptomics analysis of monolayer and neurospheres from established glioblastoma cell lines JF - Cancers N2 - Altered metabolic processes contribute to carcinogenesis by modulating proliferation, survival and differentiation. Tumours are composed of different cell populations, with cancer stem-like cells being one of the most prominent examples. This specific pool of cells is thought to be responsible for cancer growth and recurrence and plays a particularly relevant role in glioblastoma (GBM), the most lethal form of primary brain tumours. Here, we have analysed the transcriptome and metabolome of an established GBM cell line (U87) and a patient-derived GBM stem-like cell line (NCH644) exposed to neurosphere or monolayer culture conditions. By integrating transcriptome and metabolome data, we identified key metabolic pathways and gene signatures that are associated with stem-like and differentiated states in GBM cells, and demonstrated that neurospheres and monolayer cells differ substantially in their metabolism and gene regulation. Furthermore, arginine biosynthesis was identified as the most significantly regulated pathway in neurospheres, although individual nodes of this pathway were distinctly regulated in the two cellular systems. Neurosphere conditions, as opposed to monolayer conditions, cause a transcriptomic and metabolic rewiring that may be crucial for the regulation of stem-like features, where arginine biosynthesis may be a key metabolic pathway. Additionally, TCGA data from GBM patients showed significant regulation of specific components of the arginine biosynthesis pathway, providing further evidence for the importance of this metabolic pathway in GBM. KW - glioblastoma KW - neurospheres KW - monolayer KW - metabolome KW - transcriptome KW - arginine metabolism Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234110 SN - 2072-6694 VL - 13 IS - 6 ER - TY - JOUR A1 - Peck, Barrie A1 - Schug, Zachary T. A1 - Zhang, Qifeng A1 - Dankworth, Beatrice A1 - Jones, Dylan T. A1 - Smethurst, Elizabeth A1 - Patel, Rachana A1 - Mason, Susan A1 - Jian, Ming A1 - Saunders, Rebecca A1 - Howell, Michael A1 - Mitter, Richard A1 - Spencer-Dene, Bradley A1 - Stamp, Gordon A1 - McGarry, Lynn A1 - James, Daniel A1 - Shanks, Emma A1 - Aboagye, Eric O. A1 - Critchlow, Susan E. A1 - Leung, Hing Y. A1 - Harris, Adrian L. A1 - Wakelam, Michael J. O. A1 - Gottlieb, Eyal A1 - Schulze, Almut T1 - Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments JF - Cancer & Metabolism N2 - Background Enhanced macromolecule biosynthesis is integral to growth and proliferation of cancer cells. Lipid biosynthesis has been predicted to be an essential process in cancer cells. However, it is unclear which enzymes within this pathway offer the best selectivity for cancer cells and could be suitable therapeutic targets. Results Using functional genomics, we identified stearoyl-CoA desaturase (SCD), an enzyme that controls synthesis of unsaturated fatty acids, as essential in breast and prostate cancer cells. SCD inhibition altered cellular lipid composition and impeded cell viability in the absence of exogenous lipids. SCD inhibition also altered cardiolipin composition, leading to the release of cytochrome C and induction of apoptosis. Furthermore, SCD was required for the generation of poly-unsaturated lipids in cancer cells grown in spheroid cultures, which resemble those found in tumour tissue. We also found that SCD mRNA and protein expression is elevated in human breast cancers and predicts poor survival in high-grade tumours. Finally, silencing of SCD in prostate orthografts efficiently blocked tumour growth and significantly increased animal survival. Conclusions Our data implicate lipid desaturation as an essential process for cancer cell survival and suggest that targeting SCD could efficiently limit tumour expansion, especially under the metabolically compromised conditions of the tumour microenvironment. KW - SCD KW - lipidomics KW - prostate cancer KW - breast cancer KW - lipid desaturation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145905 VL - 4 IS - 6 ER - TY - JOUR A1 - Oelschlaegel, Diana A1 - Weiss Sadan, Tommy A1 - Salpeter, Seth A1 - Krug, Sebastian A1 - Blum, Galia A1 - Schmitz, Werner A1 - Schulze, Almut A1 - Michl, Patrick T1 - Cathepsin inhibition modulates metabolism and polarization of tumor-associated macrophages JF - Cancers N2 - Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH\(_2\), led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE\(_2\), which correlated with the upregulation of numerous NF\(_k\)B-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu. KW - cathepsin KW - activity-based probes KW - tumor-associated macrophage KW - autophagy KW - lysosome KW - lipid metabolism KW - inflammation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213040 SN - 2072-6694 VL - 12 IS - 9 ER - TY - JOUR A1 - Mayer, Alexander E. A1 - Löffler, Mona C. A1 - Loza Valdés, Angel E. A1 - Schmitz, Werner A1 - El-Merahbi, Rabih A1 - Trujillo-Viera, Jonathan A1 - Erk, Manuela A1 - Zhang, Thianzhou A1 - Braun, Ursula A1 - Heikenwalder, Mathias A1 - Leitges, Michael A1 - Schulze, Almut A1 - Sumara, Grzegorz T1 - The kinase PKD3 provides negative feedback on cholesterol and triglyceride synthesis by suppressing insulin signaling JF - Science Signaling N2 - Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity. KW - Protein kinase D3 (PKD3) KW - cholesterol KW - diacylglycerol (DAG) KW - liver KW - metabolism Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250025 ET - accepted manuscript ER - TY - JOUR A1 - Löhr, Mario A1 - Härtig, Wolfgang A1 - Schulze, Almut A1 - Kroiß, Matthias A1 - Sbiera, Silviu A1 - Lapa, Constantin A1 - Mages, Bianca A1 - Strobel, Sabrina A1 - Hundt, Jennifer Elisabeth A1 - Bohnert, Simone A1 - Kircher, Stefan A1 - Janaki-Raman, Sudha A1 - Monoranu, Camelia-Maria T1 - SOAT1: A suitable target for therapy in high-grade astrocytic glioma? JF - International Journal of Molecular Sciences N2 - Targeting molecular alterations as an effective treatment for isocitrate dehydrogenase-wildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages. KW - SOAT1 KW - glioblastoma KW - astrocytoma KW - IDH1/2 KW - lipid droplets KW - mitotane KW - targeted therapy Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284178 SN - 1422-0067 VL - 23 IS - 7 ER - TY - INPR A1 - Löffler, Mona C. A1 - Mayer, Alexander E. A1 - Trujillo Viera, Jonathan A1 - Loza Valdes, Angel A1 - El-Merahib, Rabih A1 - Ade, Carsten P. A1 - Karwen, Till A1 - Schmitz, Werner A1 - Slotta, Anja A1 - Erk, Manuela A1 - Janaki-Raman, Sudha A1 - Matesanz, Nuria A1 - Torres, Jorge L. A1 - Marcos, Miguel A1 - Sabio, Guadalupe A1 - Eilers, Martin A1 - Schulze, Almut A1 - Sumara, Grzegorz T1 - Protein kinase D1 deletion in adipocytes enhances energy dissipation and protects against adiposity T2 - The EMBO Journal N2 - Nutrient overload in combination with decreased energy dissipation promotes obesity and diabetes. Obesity results in a hormonal imbalance, which among others, activates G-protein coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D1 (PKD1) is a DAG effector which integrates multiple nutritional and hormonal inputs, but its physiological role in adipocytes is unknown. Here, we show that PKD1 promotes lipogenesis and suppresses mitochondrial fragmentation, biogenesis, respiration, and energy dissipation in an AMP-activated protein kinase (AMPK)-dependent manner. Moreover, mice lacking PKD1 in adipocytes are resistant to diet-induced obesity due to elevated energy expenditure. Beiging of adipocytes promotes energy expenditure and counteracts obesity. Consistently, deletion of PKD1 promotes expression of the β3-adrenergic receptor (ADRB3) in a CCAAT/enhancerbinding protein (C/EBP)-α and δ-dependent manner, which leads to the elevated expression of beige markers in adipocytes and subcutaneous adipose tissue. Finally, deletion of PKD1 in adipocytes improves insulin sensitivity and ameliorates liver steatosis. Thus, loss of PKD1 in adipocytes increases energy dissipation by several complementary mechanisms and might represent an attractive strategy to treat obesity and its related complications. KW - AMP-activated protein kinase (AMPK) KW - Beige adipocytes KW - β3 adrenergic receptor (ADRB3) KW - C/EBP KW - Protein kinase D1 (PKD1) Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176093 ER - TY - JOUR A1 - Klein-Hessling, Stefan A1 - Muhammad, Khalid A1 - Klein, Matthias A1 - Pusch, Tobias A1 - Rudolf, Ronald A1 - Flöter, Jessica A1 - Qureischi, Musga A1 - Beilhack, Andreas A1 - Vaeth, Martin A1 - Kummerow, Carsten A1 - Backes, Christian A1 - Schoppmeyer, Rouven A1 - Hahn, Ulrike A1 - Hoth, Markus A1 - Bopp, Tobias A1 - Berberich-Siebelt, Friederike A1 - Patra, Amiya A1 - Avots, Andris A1 - Müller, Nora A1 - Schulze, Almut A1 - Serfling, Edgar T1 - NFATc1 controls the cytotoxicity of CD8\(^{+}\) T cells JF - Nature Communications N2 - Cytotoxic T lymphocytes are effector CD8\(^{+}\) T cells that eradicate infected and malignant cells. Here we show that the transcription factor NFATc1 controls the cytotoxicity of mouse cytotoxic T lymphocytes. Activation of Nfatc1\(^{-/-}\) cytotoxic T lymphocytes showed a defective cytoskeleton organization and recruitment of cytosolic organelles to immunological synapses. These cells have reduced cytotoxicity against tumor cells, and mice with NFATc1-deficient T cells are defective in controlling Listeria infection. Transcriptome analysis shows diminished RNA levels of numerous genes in Nfatc1\(^{-/-}\) CD8\(^{+}\) T cells, including Tbx21, Gzmb and genes encoding cytokines and chemokines, and genes controlling glycolysis. Nfatc1\(^{-/-}\), but not Nfatc2\(^{-/-}\) CD8\(^{+}\) T cells have an impaired metabolic switch to glycolysis, which can be restored by IL-2. Genome-wide ChIP-seq shows that NFATc1 binds many genes that control cytotoxic T lymphocyte activity. Together these data indicate that NFATc1 is an important regulator of cytotoxic T lymphocyte effector functions. KW - cytotoxic T cells KW - lymphocyte activation KW - signal transduction KW - gene regulation KW - immune cells KW - NFATc1 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170353 VL - 8 IS - 511 ER - TY - JOUR A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Maier, Carina R. A1 - Fischer, Thomas A1 - Prieto-Garcia, Cristian A1 - Baluapuri, Apoorva A1 - Schwarz, Jessica A1 - Schmitz, Werner A1 - Garrido-Rodriguez, Martin A1 - Pahor, Nikolett A1 - Davies, Clare C. A1 - Bassermann, Florian A1 - Orian, Amir A1 - Wolf, Elmar A1 - Schulze, Almut A1 - Calzado, Marco A. A1 - Rosenfeldt, Mathias T. A1 - Diefenbacher, Markus E. T1 - Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease JF - Frontiers in Cell and Developmental Biology N2 - Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research. KW - non-small cell lung cancer KW - CRISPR-Cas9 KW - mouse model KW - lung cancer KW - MYC KW - JUN Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230949 SN - 2296-634X VL - 9 ER - TY - JOUR A1 - Gatto, Francesco A1 - Schulze, Almut A1 - Nielsen, Jens T1 - Systematic Analysis Reveals that Cancer Mutations Converge on Deregulated Metabolism of Arachidonate and Xenobiotics JF - Cell Reports N2 - Mutations are the basis of the clonal evolution of most cancers. Nevertheless, a systematic analysis of whether mutations are selected in cancer because they lead to the deregulation of specific biological processes independent of the type of cancer is still lacking. In this study, we correlated the genome and transcriptome of 1,082 tumors. We found that nine commonly mutated genes correlated with substantial changes in gene expression, which primarily converged on metabolism. Further network analyses circumscribed the convergence to a network of reactions, termed AraX, that involves the glutathione- and oxygen-mediated metabolism of arachidonic acid and xenobiotics. In an independent cohort of 4,462 samples, all nine mutated genes were consistently correlated with the deregulation of AraX. Among all of the metabolic pathways, AraX deregulation represented the strongest predictor of patient survival. These findings suggest that oncogenic mutations drive a selection process that converges on the deregulation of the AraX network. KW - Cancer genetics KW - Genetics research Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164814 VL - 16 IS - 3 ER - TY - JOUR A1 - De Lira, Maria Nathalia A1 - Raman, Sudha Janaki A1 - Schulze, Almut A1 - Schneider-Schaulies, Sibylle A1 - Avota, Elita T1 - Neutral Sphingomyelinase-2 (NSM 2) Controls T Cell Metabolic Homeostasis and Reprogramming During Activation JF - Frontiers in Molecular Biosciences N2 - Neutral sphingomyelinase-2 (NSM2) is a member of a superfamily of enzymes responsible for conversion of sphingomyelin into phosphocholine and ceramide at the cytosolic leaflet of the plasma membrane. Upon specific ablation of NSM2, T cells proved to be hyper-responsive to CD3/CD28 co-stimulation, indicating that the enzyme acts to dampen early overshooting activation of these cells. It remained unclear whether hyper-reactivity of NSM2-deficient T cells is supported by a deregulated metabolic activity in these cells. Here, we demonstrate that ablation of NSM2 activity affects metabolism of the quiescent CD4\(^+\) T cells which accumulate ATP in mitochondria and increase basal glycolytic activity. This supports enhanced production of total ATP and metabolic switch early after TCR/CD28 stimulation. Most interestingly, increased metabolic activity in resting NSM2-deficient T cells does not support sustained response upon stimulation. While elevated under steady-state conditions in NSM2-deficient CD4\(^+\) T cells, the mTORC1 pathway regulating mitochondria size, oxidative phosphorylation, and ATP production is impaired after 24 h of stimulation. Taken together, the absence of NSM2 promotes a hyperactive metabolic state in unstimulated CD4\(^+\) T cells yet fails to support sustained T cell responses upon antigenic stimulation. KW - neutral sphingomyelinase-2 KW - T cell receptor KW - Seahorse XF KW - oxidative phosphorylation KW - ATP-adenosine triphosphate KW - Mitochondria Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-211311 SN - 2296-889X VL - 7 ER - TY - JOUR A1 - Bensaad, Karim A1 - Favaro, Elena A1 - Lewis, Caroline A. A1 - Peck, Barrie A1 - Lord, Simon A1 - Collins, Jennifer M. A1 - Pinnick, Katherine E. A1 - Wigfield, Simon A1 - Buffa, Francesca M. A1 - Li, Ji-Liang A1 - Zhang, Qifeng A1 - Wakelam, Michael J. O. A1 - Karpe, Fredrik A1 - Schulze, Almut A1 - Harris, Adrian L. T1 - Fatty Acid Uptake and Lipid Storage Induced by HIF-1 alpha Contribute to Cell Growth and Survival after Hypoxia-Reoxygenation JF - Cell Reports N2 - An in vivo model of antiangiogenic therapy allowed us to identify genes upregulated by bevacizumab treatment, including Fatty Acid Binding Protein 3 (FABP3) and FABP7, both of which are involved in fatty acid uptake. In vitro, both were induced by hypoxia in a hypoxia-inducible factor-1 alpha (HIF-1 alpha)-dependent manner. There was a significant lipid droplet (LD) accumulation in hypoxia that was time and O-2 concentration dependent. Knockdown of endogenous expression of FABP3, FABP7, or Adipophilin (an essential LD structural component) significantly impaired LD formation under hypoxia. We showed that LD accumulation is due to FABP3/7-dependent fatty acid uptake while de novo fatty acid synthesis is repressed in hypoxia. We also showed that ATP production occurs via beta-oxidation or glycogen degradation in a cell-type-dependent manner in hypoxia-reoxygenation. Finally, inhibition of lipid storage reduced protection against reactive oxygen species toxicity, decreased the survival of cells subjected to hypoxia-reoxygenation in vitro, and strongly impaired tumorigenesis in vivo. KW - inducible factor-I KW - binding protein KW - triglyceride accumulation KW - cancer cell KW - complex-III KW - beta-oxidation KW - metabolism KW - lipogenesis KW - proliferation KW - resistance Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115162 SN - 2211-1247 VL - 9 IS - 1 ER - TY - JOUR A1 - Bartmann, Catharina A1 - Janaki Raman, Sudha R. A1 - Flöter, Jessica A1 - Schulze, Almut A1 - Bahlke, Katrin A1 - Willingstorfer, Jana A1 - Strunz, Maria A1 - Wöckel, Achim A1 - Klement, Rainer J. A1 - Kapp, Michaela A1 - Djuzenova, Cholpon S. A1 - Otto, Christoph A1 - Kämmerer, Ulrike T1 - Beta-hydroxybutyrate (3-OHB) can influence the energetic phenotype of breast cancer cells, but does not impact their proliferation and the response to chemotherapy or radiation JF - Cancer & Metabolism N2 - Background: Ketogenic diets (KDs) or short-term fasting are popular trends amongst supportive approaches for cancer patients. Beta-hydroxybutyrate (3-OHB) is the main physiological ketone body, whose concentration can reach plasma levels of 2–6 mM during KDs or fasting. The impact of 3-OHB on the biology of tumor cells described so far is contradictory. Therefore, we investigated the effect of a physiological concentration of 3 mM 3-OHB on metabolism, proliferation, and viability of breast cancer (BC) cells in vitro. Methods: Seven different human BC cell lines (BT20, BT474, HBL100, MCF-7, MDA-MB 231, MDA-MB 468, and T47D) were cultured in medium with 5 mM glucose in the presence of 3 mM 3-OHB at mild hypoxia (5% oxygen) or normoxia (21% oxygen). Metabolic profiling was performed by quantification of the turnover of glucose, lactate, and 3-OHB and by Seahorse metabolic flux analysis. Expression of key enzymes of ketolysis as well as the main monocarboxylic acid transporter MCT2 and the glucose-transporter GLUT1 was analyzed by RT-qPCR and Western blotting. The effect of 3-OHB on short- and long-term cell proliferation as well as chemo- and radiosensitivity were also analyzed. Results: 3-OHB significantly changed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in BT20 cells resulting in a more oxidative energetic phenotype. MCF-7 and MDA-MB 468 cells had increased ECAR only in response to 3-OHB, while the other three cell types remained uninfluenced. All cells expressed MCT2 and GLUT1, thus being able to uptake the metabolites. The consumption of 3-OHB was not strongly linked to mRNA overexpression of key enzymes of ketolysis and did not correlate with lactate production and glucose consumption. Neither 3-OHB nor acetoacetate did interfere with proliferation. Further, 3-OHB incubation did not modify the response of the tested BC cell lines to chemotherapy or radiation. Conclusions: We found that a physiological level of 3-OHB can change the energetic profile of some BC cell lines. However, 3-OHB failed to influence different biologic processes in these cells, e.g., cell proliferation and the response to common breast cancer chemotherapy and radiotherapy. Thus, we have no evidence that 3-OHB generally influences the biology of breast cancer cells in vitro. KW - ketogenic diet KW - β-Hydroxybutyrate KW - ketone bodies KW - breast cancer KW - seahorse KW - metabolic profile KW - chemotherapy KW - ionizing radiation Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175607 VL - 6 IS - 8 ER - TY - JOUR A1 - Bartel, Karin A1 - Pein, Helmut A1 - Popper, Bastian A1 - Schmitt, Sabine A1 - Janaki-Raman, Sudha A1 - Schulze, Almut A1 - Lengauer, Florian A1 - Koeberle, Andreas A1 - Werz, Oliver A1 - Zischka, Hans A1 - Müller, Rolf A1 - Vollmar, Angelika M. A1 - Schwarzenberg, Karin von T1 - Connecting lysosomes and mitochondria – a novel role for lipid metabolism in cancer cell death JF - Cell Communication and Signaling N2 - Background The understanding of lysosomes has been expanded in recent research way beyond their view as cellular trash can. Lysosomes are pivotal in regulating metabolism, endocytosis and autophagy and are implicated in cancer. Recently it was discovered that the lysosomal V-ATPase, which is known to induce apoptosis, interferes with lipid metabolism in cancer, yet the interplay between these organelles is poorly understood. Methods LC-MS/MS analysis was performed to investigate lipid distribution in cells. Cell survival and signaling pathways were analyzed by means of cell biological methods (qPCR, Western Blot, flow cytometry, CellTiter-Blue). Mitochondrial structure was analyzed by confocal imaging and electron microscopy, their function was determined by flow cytometry and seahorse measurements. Results Our data reveal that interfering with lysosomal function changes composition and subcellular localization of triacylglycerids accompanied by an upregulation of PGC1α and PPARα expression, master regulators of energy and lipid metabolism. Furthermore, cardiolipin content is reduced driving mitochondria into fission, accompanied by a loss of membrane potential and reduction in oxidative capacity, which leads to a deregulation in cellular ROS and induction of mitochondria-driven apoptosis. Additionally, cells undergo a metabolic shift to glutamine dependency, correlated with the fission phenotype and sensitivity to lysosomal inhibition, most prominent in Ras mutated cells. Conclusion This study sheds mechanistic light on a largely uninvestigated triangle between lysosomes, lipid metabolism and mitochondrial function. Insight into this organelle crosstalk increases our understanding of mitochondria-driven cell death. Our findings furthermore provide a first hint on a connection of Ras pathway mutations and sensitivity towards lysosomal inhibitors. KW - lysosome KW - V-ATPase KW - mitochondria KW - fission KW - apoptosis KW - lipid metabolism KW - cardiolipin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221524 VL - 17 ER -