TY - JOUR A1 - Werner, Rudolf A1 - Schmid, Jan-Stefan A1 - Higuchi, Takahiro A1 - Javadi, Mehrbod S. A1 - Rowe, Steven P. A1 - Märkl, Bruno A1 - Aulmann, Christoph A1 - Fassnacht, Martin A1 - Kroiß, Matthias A1 - Reiners, Christoph A1 - Buck, Andreas A1 - Kreissl, Michael A1 - Lapa, Constantin T1 - Predictive value of \(^{18}\)F-FDG PET in patients with advanced medullary thyroid carcinoma treated with vandetanib JF - Journal of Nuclear Medicine N2 - Introduction: Therapeutic options in advanced medullary thyroid carcinoma (MTC) have markedly improved since the introduction of tyrosine kinase inhibitors (TKI). We aimed to assess the role of metabolic imaging using 2-deoxy-2-(\(^{18}\)F)fluoro-D-glucose (\(^{18}\)F-FDG) positron emission tomography/computed tomography (PET/CT) shortly before and 3 months after initiation of TKI treatment. Methods: Eighteen patients with advanced and progressive MTC scheduled for vandetanib treatment underwent baseline \(^{18}\)F-FDG PET/CT prior to and 3 months after TKI treatment initiation. During follow-up, CT scans were performed every 3 months and analyzed according to Response Evaluation Criteria In Solid Tumors (RECIST). The predictive value for estimating progression-free (PFS) and overall survival (OS) was examined by investigating \(^{18}\)F-FDG mean/maximum standardized uptake values (SUVmean/max) of the metabolically most active lesion as well as by analyzing clinical parameters (tumor marker doubling times {calcitonin, carcinoembryonic antigen (CEA)}, prior therapies, RET (rearranged during transfection) mutational status, and disease type). Results: Within a median follow-up of 5.2 years, 9 patients experienced disease progression after a median time interval of 2.1y whereas the remainder had ongoing disease control (n=5 partial response and n=4 stable disease). Eight of the 9 patients with progressive disease died from MTC after a median of 3.5y after TKI initiation. Pre-therapeutic SUVmean >4.0 predicted a significantly shorter PFS (PFS: 1.9y vs. 5.2y; p=0.04). Furthermore, sustained high 18F-FDG uptake at 3 months with a SUVmean>2.8 tended to portend an unfavorable prognosis with a PFS of 1.9y (vs. 3.5y; p=0.3). Prolonged CEA doubling times were significantly correlated with longer PFS (r=0.7) and OS (r=0.76, p<0.01, respectively). None of the other clinical parameters had prognostic significance. Conclusions: Pre-therapeutic \(^{18}\)F-FDG PET/CT holds prognostic information in patients with advanced MTC scheduled for treatment with the TKI vandetanib. Low tumor metabolism of SUVmean < 4.0 prior to treatment predicts longer progression-free survival. KW - positron emission tomography KW - Medullärer Schilddrüsenkrebs KW - Positronen-Emissions-Tomografie KW - medullary thyroid carcinoma KW - tyrosine kinase inhibitor KW - vandetanib KW - 2- deoxy-2-(18F)fluoro-D-glucose KW - 18F-FDG Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-161256 SN - 0161-5505 N1 - This research was originally published in JNM. Rudolf A. Werner, Jan-Stefan Schmid, Takahiro Higuchi, Mehrbod S. Javadi, Steven P. Rowe, Bruno Märkl, Christoph Aulmann, Martin Fassnacht, Matthias Kroiss, Christoph Reiners, Andreas K. Buck, Michael C. Kreissl, Constantin Lapa. Predictive value of 18F-FDG PET in patients with advanced medullary thyroid carcinoma treated with vandetanib. J Nucl Med. May 1, 2018;vol. 59 no. 5: 756-761. © SNMMI. 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 - THES A1 - Kroiß, Matthias T1 - Die subzelluläre Verteilung des Regulatorproteins RS1 in Nierenepithelzellen T1 - The sucbcellular distribution of the regulatory protein RS1 in renal epithelial cells N2 - Diese Arbeit bedient sich der Immunfluoreszenzmikroskopie, um die intrazelluläre Lokalisation des mit der Plasmamembran assoziierten Regulatorproteins RS1 und eines seiner Zielproteine, des Natrium-D-Glucose-Kotransporters SGLT1, in Zellkulturmodellen des Nierenepithels (LLC-PK1- und HEK293-Zellen) zu untersuchen. Zwei polyklonale Antikörper gegen das RS1-Protein des Schweins (pRS1) wurden dafür erzeugt. In Untersuchungen am konfokalen Laser-Scanning-Mikroskop fand sich pRS1 an der Plasmamembran, im Zellkern, intrazellulär an Vesikeln sowie an einem perinukleären Kompartiment. Die Lokalisation des Proteins im Kern von LLC-PK1-Zellen nahm mit zunehmender Differenzierung der Zellen ab, pRS1 wurde in differenzierten Zellen lediglich im perinukleären Kompartiment gefunden. Dieses wurde in Kolokalisationsstudien als trans-Golgi-Netzwerk (TGN) identifiziert und dort eine Kolokalisation von pRS1 mit Clathrin und Dynamin nachgewiesen. Durch Behandlung der Zellen mit Brefeldin A wurde der Verlust von pRS1 vom TGN induziert. SGLT1 wurde überwiegend in Endosomen nachgewiesen, die entlang von Microtubuli organisiert waren. Auch im trans-Golgi-Netzwerk wurde die Anwesenheit von SGLT1 gezeigt. pSGLT1 kolokalisierte dort mit Dynamin aber nicht mit Clathrin. Es wurde demonstriert, dass experimentelle Hemmung der Proteasoms die Menge an pRS1 drastisch erhöht und gegenläufig die des Natrium-D-Glucose-Kotransporter (pSGLT1) abnimmt. Die gewonnenen Daten wurden in einem hypothetischen Modell zusammengefasst, das die gezeigten Ergebnisse mit früher gewonnenen funktionellen Experimente zu einem schlüssigen Konzept zusammenführt. N2 - RS1 is a negative regulator of solute transporters such as the Na+-D-glucose cotransporter SGLT1 on the transcriptional and posttranscriptional level. RS1 has been shown to reduce SGLT1-mediated substrate uptake upon coexpression in Xenopus oocytes. This effect is paralleled by decreased membrane surface area and can be counteracted by coexpressed dominant negative dynamin. In this study, I used immunofluorescence microscopy to determine the subcellular distribution of RS1 and SGLT1 in two renal epithelial cell lines (HEK293, LLC-PK1) that express RS1 and SGLT1 endogenously. I found that RS1 was present i) at the plasma membrane, ii) within the nucleus, iii) in small vesicles and iv) at the trans-Golgi network (TGN). At the TGN, RS1 was colocalized with clathrin and dynamin. Incubation of cells with brefeldin A abolished the association of RS1 with the TGN. SGLT1 was likewise present at the TGN although the majority of SGLT1 resided in elongated endosomes. The presence of both RS1 and SGLT1 at the TGN suggests that their functional interaction may involve adaptor proteins such as the Golgi localized, gamma-ear-containing, Arf binding (GGA) proteins. Indeed, RS1 contains a unique acidic cluster dileucine motif on its ubiquitin binding associated (UBA) domain, which we found by 3D modeling to be exposed to the surface of RS1. Taken together with previous data, this study leads to a testable model of RS1 function on the posttranscriptional level. KW - Glucose KW - Transporter KW - Regulation KW - Niere KW - Zellkultur KW - glucose KW - transporter KW - regulation KW - kidney KW - cell culture Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-20712 ER - TY - THES A1 - Kroiß, Matthias T1 - Reinigung und funktionelle Charakterisierung des SMN-Komplexes von Drosophila melanogaster T1 - Purification and functional characterization of the SMN-complex in Drosophila melanogaster N2 - Die Zusammenlageurng spleißosomaler UsnRNPs erfolgt beim Menschen und anderen Vertebraten durch den makromolekularen SMN-Komplex. Dieser besteht aus insgesamt neun Proteinen, genannt SMN und Gemin2-8. In dieser Arbeit wurde die Evolution dieser molekularen Maschine untersucht. Dazu wurden die Genome mehrerer Modellorganismen bioinformatisch nach Orthologen von SMN und seinen Komplexpartnern durchsucht. Es zeigte sich, dass SMN und Gemin2 die Kernkomponenten des Komplexes darstellen. Von diesen ausgehend kamen weitere Komponenten im Laufe der Evolution hinzu und zwar blockweise, wie es ihrer physischen Assoziation im humanen Komplex entspricht. Um diese Befunde einer biochemischen Überprüfung zu unterziehen, wurde ein neues Affinitätsepitop, das TagIt-Epitop, entwickelt. Nach stabiler Transfektion von Drosophila Schneider2-Zellen konnte das Fusionsprotein effizient exprimiert und der Drosophila-SMN-Komplex nativ aufgereinigt werden. Die massenspektrometrische Untersuchung des Komplexes zeigte, dass SMN und Gemin2 seine einzigen stöchiometrischen Komponenten sind. Dies ist in eindrucksvoller Übereinstimmung mit den bioinformatischen Daten. Der aufgereinigte Komplex lagert in vitro Sm-Proteine mit der entsprechenden UsnRNA zum UsnRNP-core-Komplex zusammen. Diese Ergebnisse ließen sich nach rekombinanter Rekonstitution des SMN/Gemin2-Dimers rekapitulieren. Dabei zeigte sich, dass der SMN-Komplex die unkoordinierte Bindung der Sm-Proteine an „falsche“ RNAs verhindert. Folglich genügen SMN und Gemin2 zur Zusammenlagerung des Sm-core-Komplexes, während die übrigen Gemine weitere Funktionen im Kontext der UsnRNP-Biogenese spielen könnten. Aus evolutionsbiologischer Sichtweise ist der SMN-Komplex aus Drosophila ein eindrückliches Beispiel, wie die Vereinfachung eines biochemischen Prozesses zur Kompaktierung des Genoms beitragen kann. N2 - In vertebrates, assembly of spliceosomal UsnRNPs is mediated by the SMN-complex, a macromolecular entity composed of the proteins SMN and Gemins 2-8. In this study, the evolution of this machinery has been investigated using complete genome assemblies of multiple model organisms. The SMN-complex has gained complexity in evolution by a block-wise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to higher complexity in metazoans, orthologs of most Gemins are missing in dipterans. In order to challenge these findings by biochemical means, I have developed a novel affinity epitope suitable for use in transfected Drosophila Schneider2-cells. Using protein mass spectrometry, the composition of the Drosophila SMN-complex has been determined. In accordance with the bioinformatic data, it consists of the core components SMN and Gemin2 only. Purified complex mediates assembly of UsnRNP core complexes in a manner very similar to its vertebrate counterpart. These results were recapitulated after recombinant reconstitution of the dSMN/dGemin2-dimer, demonstrating that the Drosophila complex also prevents mis-assembly of Smproteins onto non-target RNAs. Hence, only a minority of Gemins is required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. From a more general point of view, the evolution of the SMN-complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction. KW - Taufliege KW - Epitop KW - Antikörper KW - Biochemische Evolution KW - SMN-Komplex KW - Spinale Muskelatrophie KW - Affinitätsreinigung KW - Epitop-Tag KW - SMN-complex KW - spinal muscular atrophy KW - affinity purification KW - epitope tagging Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-28840 ER -