@article{LamatschTrifonovSchoriesetal.2011, author = {Lamatsch, D. K. and Trifonov, V. and Schories, S. and Epplen, J. T. and Schmid, M. and Schartl, M.}, title = {Isolation of a Cancer-Associated Microchromosome in the Sperm-Dependent Parthenogen Poecilia formosa}, series = {Cytogenetic and Genome Research}, volume = {135}, journal = {Cytogenetic and Genome Research}, number = {2}, issn = {1424-8581}, doi = {10.1159/000331271}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-196785}, pages = {135-142}, year = {2011}, abstract = {In the asexual all-female fish species Poecilia formosa, the Amazon molly, supernumerary chromosomes have frequently been found in both laboratory-reared and wild-caught individuals. While wild-caught individuals with B chromosomes are phenotypically indifferent from conspecifics, individuals carrying B chromosomes from recent introgression events in the laboratory show phenotypic changes. Former analyses showed that the expression of a pigment cell locus is associated with the presence of these B chromosomes. In addition, they contain a so far unidentified locus that confers a higher susceptibility to tumor formation in the presence of pigmentation pattern. Isolation by microdissection and hybridization to metaphase chromosomes revealed that they contain one or several sequences with similarity to a highly repetitive pericentromeric and subtelomeric sequence in A chromosomes. Isolation of one particular sequence by AFLP showed that the B chromosomes contain at least 1 copy of an A-chromosomal region which is highly conserved in the whole genus Poecilia, i.e. more than 5 million years old. We propose it to be a single copy sequence.}, language = {en} } @article{OnoSonoyamaNemaetal.2014, author = {Ono, Mitsuaki and Sonoyama, Wataru and Nema, Kazuki and Hara, Emilio Satoshi and Oida, Yasutaka and Pham, Hai Thanh and Yamamoto, Katushi and Hirota, Kazuo and Sugama, Kazushige and Sebald, Walter and Kuboki, Takuo}, title = {Regeneration of calvarial defects with Escherichia coli-derived rhBMP-2 adsorbed in PLGA membrane}, series = {Cells Tissues Organs}, volume = {198}, journal = {Cells Tissues Organs}, number = {5}, issn = {1422-6405}, doi = {10.1159/000356947}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-196680}, pages = {367 -- 376}, year = {2014}, abstract = {Objective: Escherichia coli-derived recombinant human bone morphogenetic protein-2 (E-BMP-2) has been shown to be as effective as mammalian cell-derived BMP-2. However, several in vitro and in vivo experiments are still necessary to validate the effectiveness of E-BMP-2 due to the difference in synthesis process, mainly related to protein nonglycosylation. The objective of this study was to investigate whether biodegradable polylactide-co-glycolide (PLGA) membrane is a suitable carrier for E-BMP-2 delivery for bone regeneration of critical-sized defects in rat calvaria. Materials and Methods: First, the osteoinductive effect of E-BMP-2 was confirmed in vitro in mouse bone marrow stromal cells by analysis of osteocalcin mRNA levels, and calcium deposition was detected by alizarin red staining. Before in vivo experiments, the release profile of E-BMP-2 from PLGA membranes was determined by ELISA. E-BMP-2 (0, 1, 5 and 10 μg/μl) was applied for ectopic and orthotopic bone formation and was analyzed by X-ray, micro-CT and histology. Results: Release-profile testing showed that PLGA membrane could retain 94\% of the initially applied E-BMP-2. Ectopic bone formation assay revealed that combination of E-BMP-2/PLGA membrane strongly induced bone formation. Stronger osteoinductivity with complete repair of critical-sized defects was observed only with PLGA membranes adsorbed with 5 and 10 μg/μl of E-BMP-2, whereas no bone formation was observed in the groups that received no membrane or 0-μg/μl dose of E-BMP-2. Conclusion: PLGA membrane was shown to be a suitable carrier for sustained release of E-BMP-2, and the E-BMP-2/PLGA membrane combination was demonstrated to be efficient in bone regeneration in a model of critical-sized defects.}, language = {en} } @phdthesis{Gruendl2021, author = {Gr{\"u}ndl, Marco}, title = {Biochemical characterization of the MMB-Hippo crosstalk and its physiological relevance for heart development}, doi = {10.25972/OPUS-21332}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213328}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The Myb-MuvB (MMB) complex plays an essential role in the time-dependent transcriptional activation of mitotic genes. Recently, our laboratory identified a novel crosstalk between the MMB-complex and YAP, the transcriptional coactivator of the Hippo pathway, to coregulate a subset of mitotic genes (Pattschull et al., 2019). Several genetic studies have shown that the Hippo-YAP pathway is essential to drive cardiomyocyte proliferation during cardiac development (von Gise et al., 2012; Heallen et al., 2011; Xin et al., 2011). However, the exact mechanisms of how YAP activates proliferation of cardiomyocytes is not known. This doctoral thesis addresses the physiological role of the MMB-Hippo crosstalk within the heart and characterizes the YAP-B-MYB interaction with the overall aim to identify a potent inhibitor of YAP. The results reported in this thesis indicate that complete loss of the MMB scaffold protein LIN9 in heart progenitor cells results in thinning of ventricular walls, reduced cardiomyocyte proliferation and early embryonic lethality. Moreover, genetic experiments using mice deficient in SAV1, a core component of the Hippo pathway, and LIN9-deficient mice revealed that the correct function of the MMB complex is critical for proliferation of cardiomyocytes due to Hippo-deficiency. Whole genome transcriptome profiling as well as genome wide binding studies identified a subset of Hippo-regulated cell cycle genes as direct targets of MMB. By proximity ligation assay (PLA), YAP and B-MYB were discovered to interact in embryonal cardiomyocytes. Biochemical approaches, such as co-immunoprecipitation assays, GST-pulldown assays, and µSPOT-based peptide arrays were employed to characterize the YAP-B-MYB interaction. Here, a PY motif within the N-terminus of B-MYB was found to directly interact with the YAP WW-domains. Consequently, the YAP WW-domains were important for the ability of YAP to drive proliferation in cardiomyocytes and to activate MMB target genes in differentiated C2C12 cells. The biochemical information obtained from the interaction studies was utilized to develop a novel competitive inhibitor of YAP called MY-COMP (Myb-YAP competition). In MY-COMP, the protein fragment of B-MYB containing the YAP binding domain is fused to a nuclear localization signal. Co-immunoprecipitation studies as well as PLA revealed that the YAP-B-MYB interaction is robustly blocked by expression of MY-COMP. Adenoviral overexpression of MY-COMP in embryonal cardiomyocytes suppressed entry into mitosis and blocked the pro-proliferative function of YAP. Strikingly, characterization of the cellular phenotype showed that ectopic expression of MY-COMP led to growth defects, nuclear abnormalities and polyploidization in HeLa cells. Taken together, the results of this thesis reveal the mechanism of the crosstalk between the Hippo signaling pathway and the MMB complex in the heart and form the basis for interference with the oncogenic activity of the Hippo coactivator YAP.}, subject = {Zellzyklus}, language = {en} } @phdthesis{Simon2019, author = {Simon, Katja}, title = {Identifying the role of Myb-MuvB in gene expression and proliferation of lung cancer cells}, doi = {10.25972/OPUS-16181}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161814}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The evolutionary conserved Myb-MuvB (MMB) multiprotein complex is a transcriptional master regulator of mitotic gene expression. The MMB subunits B-MYB, FOXM1 as well as target genes of MMB are often overexpressed in different cancer types. Elevated expression of these genes correlates with an advanced tumor state and a poor prognosis for patients. Furthermore, it has been reported that pathways, which are involved in regulating the mitotic machinery are attractive for a potential treatment of cancers harbouring Ras mutations (Luo et al., 2009). This suggest that the MMB complex could be required for tumorigenesis by mediating overactivity of mitotic genes and that the MMB could be a useful target for lung cancer treatment. However, although MMB has been characterized biochemically, the contribution of MMB to tumorigenesis is largely unknown in particular in vivo. In this thesis, it was demonstrated that the MMB complex is required for lung tumorigenesis in vivo in a mouse model of non small cell lung cancer. Elevated levels of B-MYB, NUSAP1 or CENPF in advanced tumors as opposed to low levels of these proteins levels in grade 1 or 2 tumors support the possible contribution of MMB to lung tumorigenesis and the oncogenic potential of B-MYB.The tumor growth promoting function of B-MYB was illustrated by a lower fraction of KI-67 positive cells in vivo and a significantly high impairment in proliferation after loss of B-Myb in vitro. Defects in cytokinesis and an abnormal cell cycle profile after loss of B-Myb underscore the impact of B-MYB on proliferation of lung cancer cell lines. The incomplete recombination of B-Myb in murine lung tumors and in the tumor derived primary cell lines illustrates the selection pressure against the complete loss of B-Myb and further demonstrats that B-Myb is a tumor-essential gene. In the last part of this thesis, the contribution of MMB to the proliferation of human lung cancer cells was demonstrated by the RNAi-mediated depletion of B-Myb. Detection of elevated B-MYB levels in human adenocarcinoma and a reduced proliferation, cytokinesis defects and abnormal cell cycle profile after loss of B-MYB in human lung cancer cell lines underlines the potential of B-MYB to serve as a clinical marker.}, subject = {Lungenkrebs}, language = {en} } @article{JonesFrucianoKelleretal.2016, author = {Jones, Julia C. and Fruciano, Carmelo and Keller, Anja and Schartl, Manfred and Meyer, Axel}, title = {Evolution of the elaborate male intromittent organ of Xiphophorus fishes}, series = {Ecology and Evolution}, volume = {6}, journal = {Ecology and Evolution}, number = {20}, doi = {10.1002/ece3.2396}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164956}, pages = {7207-7220}, year = {2016}, abstract = {Internally fertilizing animals show a remarkable diversity in male genital morphology that is associated with sexual selection, and these traits are thought to be evolving particularly rapidly. Male fish in some internally fertilizing species have "gonopodia," highly modified anal fins that are putatively important for sexual selection. However, our understanding of the evolution of genital diversity remains incomplete. Contrary to the prediction that male genital traits evolve more rapidly than other traits, here we show that gonopodial traits and other nongonopodial traits exhibit similar evolutionary rates of trait change and also follow similar evolutionary models in an iconic genus of poeciliid fish (Xiphophorus spp.). Furthermore, we find that both mating and nonmating natural selection mechanisms are unlikely to be driving the diverse Xiphophorus gonopodial morphology. Putative holdfast features of the male genital organ do not appear to be influenced by water flow, a candidate selective force in aquatic habitats. Additionally, interspecific divergence in gonopodial morphology is not significantly higher between sympatric species, than between allopatric species, suggesting that male genitals have not undergone reproductive character displacement. Slower rates of evolution in gonopodial traits compared with a subset of putatively sexually selected nongenital traits suggest that different selection mechanisms may be acting on the different trait types. Further investigations of this elaborate trait are imperative to determine whether it is ultimately an important driver of speciation.}, language = {en} } @article{BrunetVolffSchartl2016, author = {Brunet, Fr{\´e}d{\´e}ric G. and Volff, Jean-Nicolas and Schartl, Manfred}, title = {Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates}, series = {Genome Biology Evolution}, volume = {8}, journal = {Genome Biology Evolution}, number = {15}, doi = {10.1093/gbe/evw103}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146988}, pages = {1600-1613}, year = {2016}, abstract = {The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75\% after 1R/2R, 64.4\% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling.}, language = {en} } @phdthesis{Jung2016, author = {Jung, Lisa Anna}, title = {Targeting MYC Function as a Strategy for Tumor Therapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146993}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {A large fraction of human tumors exhibits aberrant expression of the oncoprotein MYC. As a transcription factor regulating various cellular processes, MYC is also crucially involved in normal development. Direct targeting of MYC has been a major challenge for molecular cancer drug discovery. The proof of principle that its inhibition is nevertheless feasible came from in vivo studies using a dominant-negative allele of MYC termed OmoMYC. Systemic expression of OmoMYC triggered long-term tumor regression with mild and fully reversible side effects on normal tissues. In this study, OmoMYC's mode of action was investigated combining methods of structural biology and functional genomics to elucidate how it is able to preferentially affect oncogenic functions of MYC. The crystal structure of the OmoMYC homodimer, both in the free and the E-box-bound state, was determined, which revealed that OmoMYC forms a stable homodimer, and as such, recognizes DNA via the same base-specific DNA contacts as the MYC/MAX heterodimer. OmoMYC binds DNA with an equally high affinity as MYC/MAX complexes. RNA-sequencing showed that OmoMYC blunts both MYC-dependent transcriptional activation and repression. Genome-wide DNA-binding studies using chromatin immunoprecipitation followed by high-throughput sequencing revealed that OmoMYC competes with MYC/MAX complexes on chromatin, thereby reducing their occupancy at consensus DNA binding sites. The most prominent decrease in MYC binding was seen at low-affinity promoters, which were invaded by MYC at oncogenic levels. Strikingly, gene set enrichment analyses using OmoMYC-regulated genes enabled the identification of tumor subgroups with high MYC levels in multiple tumor entities. Together with a targeted shRNA screen, this identified novel targets for the eradication of MYC-driven tumors, such as ATAD3A, BOP1, and ADRM1. In summary, the findings suggest that OmoMYC specifically inhibits tumor cell growth by attenuating the expression of rate-limiting proteins in cellular processes that respond to elevated levels of MYC protein using a DNA-competitive mechanism. This opens up novel strategies to target oncogenic MYC functions for tumor therapy.}, subject = {Myc}, language = {en} } @phdthesis{Maurus2016, author = {Maurus, Katja}, title = {Melanoma Maintenance by the AP1 Transcription Factor FOSL1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142995}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Identifying novel driver genes in cancer remains a crucial step towards development of new therapeutic approaches and the basic understanding of the disease. This work describes the impact of the AP1 transcription activator component FOSL1 on melanoma maintenance. FOSL1 is strongly upregulated during the progression of melanoma and the protein abundance is highest in metastases. I found that the regulation of FOSL1 is strongly dependent on ERK1/2- and PI3K- signaling, two pathways frequently activated in melanoma. Moreover, the involvement of p53 in FOSL1 regulation in melanoma was investigated. Elevated levels of the tumor suppressor led to decreased FOSL1 protein levels in a miR34a/miR34c- dependent manner. The benefit of elevated FOSL1 amounts in human melanoma cell lines was analyzed by overexpression of FOSL1 in cell lines with low endogenous FOSL1 levels. Enhanced levels of FOSL1 had several pro-tumorigenic effects in human melanoma cell lines. Besides increased proliferation and migration rates, FOSL1 overexpression induced the colony forming ability of the cells. Additionally, FOSL1 was necessary for anchorage independent growth in 3D cell cultures. Microarray analyses revealed novel downstream effectors of FOSL1. On the one hand, FOSL1 was able to induce the transcription of different neuron-related genes, such as NEFL, NRP1 and TUBB3. On the other hand, FOSL1 influenced the transcription of DCT, a melanocyte specific gene, in dependence of the differentiation of the melanoma cell line, indicating dedifferentiation. Furthermore, FOSL1 induced the transcription of HMGA1, a chromatin remodeling protein with reprogramming ability, which is characteristic for stem cells. Consequently, the influence of HMGA1 on melanoma maintenance was investigated. In addition to decreased proliferation and reduced anoikis resistance, HMGA1 knockdown reduced melanoma cell survival. Interestingly, the FOSL1 induced pro-tumorigenic effects were demonstrated to be dependent on the HMGA1 level. HMGA1 manipulation reversed FOSL1 induced proliferation and colony forming ability, as well as the anchorage independent growth effect. In conclusion, I could show that additional FOSL1 confers a clear growth benefit to melanoma cells. This benefit is attributed to the induction of stem cell determinants, but can be blocked by the inhibition of the ERK1/2 or PI3K signaling pathways.}, subject = {Melanom}, language = {en} } @article{PeckSchugZhangetal.2016, author = {Peck, Barrie and Schug, Zachary T. and Zhang, Qifeng and Dankworth, Beatrice and Jones, Dylan T. and Smethurst, Elizabeth and Patel, Rachana and Mason, Susan and Jian, Ming and Saunders, Rebecca and Howell, Michael and Mitter, Richard and Spencer-Dene, Bradley and Stamp, Gordon and McGarry, Lynn and James, Daniel and Shanks, Emma and Aboagye, Eric O. and Critchlow, Susan E. and Leung, Hing Y. and Harris, Adrian L. and Wakelam, Michael J. O. and Gottlieb, Eyal and Schulze, Almut}, title = {Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments}, series = {Cancer \& Metabolism}, volume = {4}, journal = {Cancer \& Metabolism}, number = {6}, doi = {10.1186/s40170-016-0146-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145905}, year = {2016}, abstract = {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.}, language = {en} } @article{WilliamsChagtaiAlcaideGermanetal.2015, author = {Williams, Richard D. and Chagtai, Tasnim and Alcaide-German, Marisa and Apps, John and Wegert, Jenny and Popov, Sergey and Vujanic, Gordan and Van Tinteren, Harm and Van den Heuvel-Eibrink, Marry M and Kool, Marcel and De Kraker, Jan and Gisselsson, David and Graf, Norbert and Gessler, Manfred and Pritchard-Jones, Kathy}, title = {Multiple mechanisms of MYCN dysregulation in Wilms tumour}, series = {Oncotarget}, volume = {6}, journal = {Oncotarget}, number = {9}, doi = {10.18632/oncotarget.3377}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143471}, pages = {7232-7243}, year = {2015}, abstract = {Genomic gain of the proto-oncogene transcription factor gene MYCN is associated with poor prognosis in several childhood cancers. Here we present a comprehensive copy number analysis of MYCN in Wilms tumour (WT), demonstrating that gain of this gene is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. Using whole exome and gene-specific sequencing, together with methylation and expression profiling, we show that MYCN is targeted by other mechanisms, including a recurrent somatic mutation, P44L, and specific DNA hypomethylation events associated with MYCN overexpression in tumours with high risk histologies. We describe parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumours of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolve over time. We report a second bilateral case in which MYCN gain is a germline aberration. Our results suggest a significant role for MYCN dysregulation in the molecular biology of Wilms tumour. We conclude that MYCN gain is prognostically significant, and suggest that the novel P44L somatic variant is likely to be an activating mutation.}, language = {en} } @article{MatosSucenaMachadoetal.2011, author = {Matos, Isa and Sucena, {\`E}lio and Machado, Miguel P and Gardner, Rui and In{\´a}cio, {\^A}ngela and Schartl, Manfred and Coelho, Maria M}, title = {Ploidy mosaicism and allele-specific gene expression differences in the allopolyploid \(Squalius\) \(alburnoides\)}, series = {BMC Genetics}, volume = {12}, journal = {BMC Genetics}, number = {101}, doi = {10.1186/1471-2156-12-101}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142879}, pages = {1-10}, year = {2011}, abstract = {Background Squalius alburnoides is an Iberian cyprinid fish resulting from an interspecific hybridisation between Squalius pyrenaicus females (P genome) and males of an unknown Anaecypris hispanica- like species (A genome). S. alburnoides is an allopolyploid hybridogenetic complex, which makes it a likely candidate for ploidy mosaicism occurrence, and is also an interesting model to address questions about gene expression regulation and genomic interactions. Indeed, it was previously suggested that in S. alburnoides triploids (PAA composition) silencing of one of the three alleles (mainly of the P allele) occurs. However, not a whole haplome is inactivated but a more or less random inactivation of alleles varying between individuals and even between organs of the same fish was seen. In this work we intended to correlate expression differences between individuals and/or between organs to the occurrence of mosaicism, evaluating if mosaics could explain previous observations and its impact on the assessment of gene expression patterns. Results To achieve our goal, we developed flow cytometry and cell sorting protocols for this system generating more homogenous cellular and transcriptional samples. With this set-up we detected 10\% ploidy mosaicism within the S. alburnoides complex, and determined the allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin) in cells from liver and kidney of mosaic and non-mosaic individuals coming from different rivers over a wide geographic range. Conclusions Ploidy mosaicism occurs sporadically within the S. alburnoides complex, but in a frequency significantly higher than reported for other organisms. Moreover, we could exclude the influence of this phenomenon on the detection of variable allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin) in cells from liver and kidney of triploid individuals. Finally, we determined that the expression patterns previously detected only in a narrow geographic range is not a local restricted phenomenon but is pervasive in rivers where S. pyrenaicus is sympatric with S. alburnoides. We discuss mechanisms that could lead to the formation of mosaic S. alburnoides and hypothesise about a relaxation of the mechanisms that impose a tight control over mitosis and ploidy control in mixoploids."}, language = {en} } @article{KrebsSolimandoKalogirouetal.2020, author = {Krebs, Markus and Solimando, Antonio Giovanni and Kalogirou, Charis and Marquardt, Andr{\´e} and Frank, Torsten and Sokolakis, Ioannis and Hatzichristodoulou, Georgios and Kneitz, Susanne and Bargou, Ralf and K{\"u}bler, Hubert and Schilling, Bastian and Spahn, Martin and Kneitz, Burkhard}, title = {miR-221-3p Regulates VEGFR2 Expression in High-Risk Prostate Cancer and Represents an Escape Mechanism from Sunitinib In Vitro}, series = {Journal of Clinical Medicine}, volume = {9}, journal = {Journal of Clinical Medicine}, number = {3}, issn = {2077-0383}, doi = {10.3390/jcm9030670}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203168}, year = {2020}, abstract = {Downregulation of miR-221-3p expression in prostate cancer (PCa) predicted overall and cancer-specific survival of high-risk PCa patients. Apart from PCa, miR-221-3p expression levels predicted a response to tyrosine kinase inhibitors (TKI) in clear cell renal cell carcinoma (ccRCC) patients. Since this role of miR-221-3p was explained with a specific targeting of VEGFR2, we examined whether miR-221-3p regulated VEGFR2 in PCa. First, we confirmed VEGFR2/KDR as a target gene of miR-221-3p in PCa cells by applying Luciferase reporter assays and Western blotting experiments. Although VEGFR2 was mainly downregulated in the PCa cohort of the TCGA (The Cancer Genome Atlas) database, VEGFR2 was upregulated in our high-risk PCa cohort (n = 142) and predicted clinical progression. In vitro miR-221-3p acted as an escape mechanism from TKI in PC3 cells, as displayed by proliferation and apoptosis assays. Moreover, we confirmed that Sunitinib induced an interferon-related gene signature in PC3 cells by analyzing external microarray data and by demonstrating a significant upregulation of miR-221-3p/miR-222-3p after Sunitinib exposure. Our findings bear a clinical perspective for high-risk PCa patients with low miR-221-3p levels since this could predict a favorable TKI response. Apart from this therapeutic niche, we identified a partially oncogenic function of miR-221-3p as an escape mechanism from VEGFR2 inhibition.}, language = {en} } @phdthesis{Dirks2019, author = {Dirks, Johannes}, title = {Charakterisierung der Wechselwirkung zwischen N-Myc und Aurora-A im MYCN-amplifizierten Neuroblastom}, doi = {10.25972/OPUS-18660}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186600}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Im Neuroblastom ist die Amplifikation des MYCN-Gens, eines Mitglieds der MYC-Onkogenfamilie, mit einer ung{\"u}nstigen Prognose assoziiert. Der von dem Gen kodierte Transkriptionsfaktor N-Myc ist f{\"u}r die Proliferation der MYCN-amplifizierten Neuroblastomzelllinien notwendig und seine Depletion oder Destabilisierung f{\"u}hren zum Proliferationsarrest (Otto et al., 2009). Da N-Myc auf Proteinebene durch die Interaktion mit der mitotischen Kinase Aurora-A stabilisiert wird, bewirkt deren Depletion oder die Hemmung der Interaktion der beiden Proteine mittels spezieller Aurora- A-Inhibitoren (z.B. MLN8054 und MLN8237) ebenso eine Hemmung der Proliferation - in vitro und in vivo (Brockmann et al., 2013). Bisher ist jedoch unklar, {\"u}ber welchen Mechanismus Aurora-A die Stabilisierung von N-Myc erreicht, die Kinaseaktivit{\"a}t spielt hierbei jedoch keine Rolle (Otto et al., 2009). Eine M{\"o}glichkeit stellt die Rekrutierung von Usps dar, die das angeh{\"a}ngte Ubiquitinsignal so modifizieren, dass die Erkennung und der Abbau des Proteins durch das Proteasom verringert werden. In der vorliegenden Arbeit wurde die Wirkung von Usp7 und Usp11 auf die Stabilit{\"a}t von N-Myc untersucht. F{\"u}r beide konnte in Immunpr{\"a}zipitationen die Interaktion mit N-Myc gezeigt werden. Ebenso erh{\"o}hten beide Proteasen in {\"U}berexpressionsexperimenten die vorhandene Menge an NMyc. Die Depletion von Usp7 mittels shRNAs f{\"u}hrte in IMR-32 zu einem Arrest in der G1-Phase und zur Differenzierung der Zellen. Gleichzeitig wurden stark erniedrigte mRNA- und Proteinmengen von N-Myc und Aurora-A nachgewiesen. Es konnte jedoch nicht eindeutig gezeigt werden, ob die beobachteten zellul{\"a}ren Effekte durch eine vermehrte proteasomale Degradation von N-Myc begr{\"u}ndet sind oder ob dabei die ver{\"a}nderte Regulation weiterer Zielproteine von Usp7 eine Rolle spielt. Die Depletion von Usp11 mit shRNAs bewirkte eine Abnahme der N-Myc-Mengen auf posttranslationaler Ebene. Somit stellen beide Usps vielversprechende Angriffspunkte einer gezielten Therapie in MYCN-amplifizierten Neuroblastomen dar und sollten deshalb Gegenstand weiterf{\"u}hrender Untersuchungen sein. {\"U}ber welche Proteindom{\"a}ne in N-Myc die Interaktion mit Aurora-A stattfindet ist nicht bekannt. Eine m{\"o}gliche Pseudosubstratbindungssequenz in Myc-Box I (Idee Richard Bayliss, University of Leicester) wurde in der vorliegenden Arbeit untersucht. Durch Mutation dieser Sequenz sollte die Bindung von Aurora-A unm{\"o}glich gemacht werden. Allerdings wurde die erwartete Abnahme der St{\"a}rke der Interaktion von Aurora-A und N-Myc durch die Mutation ebensowenig beobachtet wie eine verringerte Stabilit{\"a}t. Die Regulation der Phosphorylierung von N-Myc im Verlauf des Zellzyklus wurde durch die Mutation beeintr{\"a}chtigt. Wie diese Ver{\"a}nderung exakt zu begr{\"u}nden ist bedarf weiterer Experimente}, subject = {Neuroblastom}, language = {de} } @article{OttoHahlbrockEichetal.2016, author = {Otto, Christoph and Hahlbrock, Theresa and Eich, Kilian and Karaaslan, Ferdi and J{\"u}rgens, Constantin and Germer, Christoph-Thomas and Wiegering, Armin and K{\"a}mmerer, Ulrike}, title = {Antiproliferative and antimetabolic effects behind the anticancer property of fermented wheat germ extract}, series = {BMC Complementary and Alternative Medicine}, volume = {16}, journal = {BMC Complementary and Alternative Medicine}, number = {160}, doi = {10.1186/s12906-016-1138-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146013}, year = {2016}, abstract = {Background Fermented wheat germ extract (FWGE) sold under the trade name Avemar exhibits anticancer activity in vitro and in vivo. Its mechanisms of action are divided into antiproliferative and antimetabolic effects. Its influcence on cancer cell metabolism needs further investigation. One objective of this study, therefore, was to further elucidate the antimetabolic action of FWGE. The anticancer compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ) is the major bioactive compound in FWGE and is probably responsible for its anticancer activity. The second objective of this study was to compare the antiproliferative properties in vitro of FWGE and the DMBQ compound. Methods The IC\(_{50}\) values of FWGE were determined for nine human cancer cell lines after 24 h of culture. The DMBQ compound was used at a concentration of 24 μmol/l, which is equal to the molar concentration of DMBQ in FWGE. Cell viability, cell cycle, cellular redox state, glucose consumption, lactic acid production, cellular ATP levels, and the NADH/NAD\(^+\) ratio were measured. Results The mean IC\(_{50}\) value of FWGE for the nine human cancer cell lines tested was 10 mg/ml. Both FWGE (10 mg/ml) and the DMBQ compound (24 μmol/l) induced massive cell damage within 24 h after starting treatment, with changes in the cellular redox state secondary to formation of intracellular reactive oxygen species. Unlike the DMBQ compound, which was only cytotoxic, FWGE exhibited cytostatic and growth delay effects in addition to cytotoxicity. Both cytostatic and growth delay effects were linked to impaired glucose utilization which influenced the cell cycle, cellular ATP levels, and the NADH/NAD\(^+\) ratio. The growth delay effect in response to FWGE treatment led to induction of autophagy. Conclusions FWGE and the DMBQ compound both induced oxidative stress-promoted cytotoxicity. In addition, FWGE exhibited cytostatic and growth delay effects associated with impaired glucose utilization which led to autophagy, a possible previously unknown mechanism behind the influence of FWGE on cancer cell metabolism.}, language = {en} } @article{KneitzMishraChalopinetal.2016, author = {Kneitz, Susanne and Mishra, Rasmi R. and Chalopin, Domitille and Postlethwait, John and Warren, Wesley C. and Walther, Ronald B. and Schartl, Manfred}, title = {Germ cell and tumor associated piRNAs in the medaka and \(Xiphophorus\) melanoma models}, series = {BMC Genomics}, volume = {17}, journal = {BMC Genomics}, number = {357}, doi = {10.1186/s12864-016-2697-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146028}, year = {2016}, abstract = {Background A growing number of studies report an abnormal expression of Piwi-interacting RNAs (piRNAs) and the piRNA processing enzyme Piwi in many cancers. Whether this finding is an epiphenomenon of the chaotic molecular biology of the fast dividing, neoplastically transformed cells or is functionally relevant to tumorigenesisis is difficult to discern at present. To better understand the role of piRNAs in cancer development small laboratory fish models can make a valuable contribution. However, little is known about piRNAs in somatic and neoplastic tissues of fish. Results To identify piRNA clusters that might be involved in melanoma pathogenesis, we use several transgenic lines of medaka, and platyfish/swordtail hybrids, which develop various types of melanoma. In these tumors Piwi, is expressed at different levels, depending on tumor type. To quantify piRNA levels, whole piRNA populations of testes and melanomas of different histotypes were sequenced. Because no reference piRNA cluster set for medaka or Xiphophorus was yet available we developed a software pipeline to detect piRNA clusters in our samples and clusters were selected that were enriched in one or more samples. We found several loci to be overexpressed or down-regulated in different melanoma subtypes as compared to hyperpigmented skin. Furthermore, cluster analysis revealed a clear distinction between testes, low-grade and high-grade malignant melanoma in medaka. Conclusions Our data imply that dysregulation of piRNA expression may be associated with development of melanoma. Our results also reinforce the importance of fish as a suitable model system to study the role of piRNAs in tumorigenesis.}, language = {en} } @article{LichthardtKerscherDietzetal.2016, author = {Lichthardt, Sven and Kerscher, Alexander and Dietz, Ulrich A. and Jurowich, Christian and Kunzmann, Volker and von Rahden, Burkhard H. A. and Germer, Christoph-Thomas and Wiegering, Armin}, title = {Original article: role of adjuvant chemotherapy in a perioperative chemotherapy regimen for gastric cancer}, series = {BMC Cancer}, volume = {16}, journal = {BMC Cancer}, number = {650}, doi = {10.1186/s12885-016-2708-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147743}, year = {2016}, abstract = {Background Multimodal treatment strategies - perioperative chemotherapy (CTx) and radical surgery - are currently accepted as treatment standard for locally advanced gastric cancer. However, the role of adjuvant postoperative CTx (postCTx) in addition to neoadjuvant preoperative CTx (preCTx) in this setting remains controversial. Methods Between 4/2006 and 12/2013, 116 patients with locally advanced gastric cancer were treated with preCTx. 72 patients (62 \%), in whom complete tumor resection (R0, subtotal/total gastrectomy with D2-lymphadenectomy) was achieved, were divided into two groups, one of which receiving adjuvant therapy (n = 52) and one without (n = 20). These groups were analyzed with regard to survival and exclusion criteria for adjuvant therapy. Results Postoperative complications, as well as their severity grade, did not correlate with fewer postCTx cycles administered (p = n.s.). Long-term survival was shorter in patients receiving postCTx in comparison to patients without postCTx, but did not show statistical significance. In per protocol analysis by excluding two patients with perioperative death, a shorter 3-year survival rate was observed in patients receiving postCTx compared to patients without postCTx (3-year survival: 71.2 \% postCTx group vs. 90.0 \% non-postCTx group; p = 0.038). Conclusion These results appear contradicting to the anticipated outcome. While speculative, they question the value of post-CTx. Prospectively randomized studies are needed to elucidate the role of postCTx.}, language = {en} } @article{KrajinovicReimerKudlichetal.2016, author = {Krajinovic, K. and Reimer, S. and Kudlich, T. and Germer, C. T. and Wiegering, A.}, title = {"Rendezvous technique" for intraluminal vacuum therapy of anastomotic leakage of the jejunum}, series = {Surgical Case Reports}, volume = {2}, journal = {Surgical Case Reports}, number = {114}, doi = {10.1186/s40792-016-0243-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147883}, year = {2016}, abstract = {Background Anastomotic leakage (AL) is one of the most common and serious complications following visceral surgery. In recent years, endoluminal vacuum therapy has dramatically changed therapeutic options for AL, but its use has been limited to areas easily accessible by endoscope. Case presentation We describe the first use of endoluminal vacuum therapy in the small intestine employing a combined surgical and endoscopic "rendezvous technique" in which the surgeon assists the endoscopic placement of an endoluminal vacuum therapy sponge in the jejunum by means of a pullback string. This technique led to a completely closed AL after 27 days and 7 changes of the endosponge. Conclusion The combined surgical and endoscopic rendezvous technique can be useful in cases of otherwise difficult endosponge placement.}, language = {en} } @article{WolterHanselmannPattschulletal.2017, author = {Wolter, Patrick and Hanselmann, Steffen and Pattschull, Grit and Schruf, Eva and Gaubatz, Stefan}, title = {Central spindle proteins and mitotic kinesins are direct transcriptional targets of MuvB, B-MYB and FOXM1 in breast cancer cell lines and are potential targets for therapy}, series = {Oncotarget}, volume = {8}, journal = {Oncotarget}, number = {7}, doi = {10.18632/oncotarget.14466}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171851}, pages = {11160-11172}, year = {2017}, abstract = {The MuvB multiprotein complex, together with B-MYB and FOXM1 (MMB-FOXM1), plays an essential role in cell cycle progression by regulating the transcription of genes required for mitosis and cytokinesis. In many tumors, B-MYB and FOXM1 are overexpressed as part of the proliferation signature. However, the transcriptional targets that are important for oncogenesis have not been identified. Given that mitotic kinesins are highly expressed in cancer cells and that selected kinesins have been reported as target genes of MMB-FOXM1, we sought to determine which mitotic kinesins are directly regulated by MMB-FOXM1. We demonstrate that six mitotic kinesins and two microtubule-associated non-motor proteins (MAPs) CEP55 and PRC1 are direct transcriptional targets of MuvB, B-MYB and FOXM1 in breast cancer cells. Suppression of KIF23 and PRC1 strongly suppressed proliferation of MDA-MB-231 cells. The set of MMB-FOXM1 regulated kinesins genes and 4 additional kinesins which we referred to as the mitotic kinesin signature (MKS) is linked to poor outcome in breast cancer patients. Thus, mitotic kinesins could be used as prognostic biomarker and could be potential therapeutic targets for the treatment of breast cancer.}, language = {en} } @article{SanderXuEilersetal.2017, author = {Sander, Bodo and Xu, Wenshan and Eilers, Martin and Popov, Nikita and Lorenz, Sonja}, title = {A conformational switch regulates the ubiquitin ligase HUWE1}, series = {eLife}, volume = {6}, journal = {eLife}, doi = {10.7554/eLife.21036}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171862}, year = {2017}, abstract = {The human ubiquitin ligase HUWE1 has key roles in tumorigenesis, yet it is unkown how its activity is regulated. We present the crystal structure of a C-terminal part of HUWE1, including the catalytic domain, and reveal an asymmetric auto-inhibited dimer. We show that HUWE1 dimerizes in solution and self-associates in cells, and that both occurs through the crystallographic dimer interface. We demonstrate that HUWE1 is inhibited in cells and that it can be activated by disruption of the dimer interface. We identify a conserved segment in HUWE1 that counteracts dimer formation by associating with the dimerization region intramolecularly. Our studies reveal, intriguingly, that the tumor suppressor p14ARF binds to this segment and may thus shift the conformational equilibrium of HUWE1 toward the inactive state. We propose a model, in which the activity of HUWE1 underlies conformational control in response to physiological cues—a mechanism that may be exploited for cancer therapy.}, language = {en} } @unpublished{LoefflerMayerTrujilloVieraetal.2018, author = {L{\"o}ffler, Mona C. and Mayer, Alexander E. and Trujillo Viera, Jonathan and Loza Valdes, Angel and El-Merahib, Rabih and Ade, Carsten P. and Karwen, Till and Schmitz, Werner and Slotta, Anja and Erk, Manuela and Janaki-Raman, Sudha and Matesanz, Nuria and Torres, Jorge L. and Marcos, Miguel and Sabio, Guadalupe and Eilers, Martin and Schulze, Almut and Sumara, Grzegorz}, title = {Protein kinase D1 deletion in adipocytes enhances energy dissipation and protects against adiposity}, series = {The EMBO Journal}, journal = {The EMBO Journal}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176093}, year = {2018}, abstract = {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.}, language = {en} }