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Redmer T.,University Hospital Berlin | Welte Y.,University Hospital Berlin | Behrens D.,Experimental Pharmacology and Oncology Berlin Buch GmbH | Fichtner I.,Experimental Pharmacology and Oncology Berlin Buch GmbH | And 6 more authors.
PLoS ONE | Year: 2014

Background: Large-scale genomic analyses of patient cohorts have revealed extensive heterogeneity between individual tumors, contributing to treatment failure and drug resistance. In malignant melanoma, heterogeneity is thought to arise as a consequence of the differentiation of melanoma-initiating cells that are defined by cell-surface markers like CD271 or CD133. Results: Here we confirmed that the nerve growth factor receptor (CD271) is a crucial determinant of tumorigenicity, stem-like properties, heterogeneity and plasticity in melanoma cells. Stable shRNA mediated knock-down of CD271 in patient-derived melanoma cells abrogated their tumor-initiating and colony-forming capacity. A genome-wide expression profiling and gene-set enrichment analysis revealed novel connections of CD271 with melanoma-associated genes like CD133 and points to a neural crest stem cell (NCSC) signature lost upon CD271 knock-down. In a meta-analysis we have determined a shared set of 271 differentially regulated genes, linking CD271 to SOX10, a marker that specifies the neural crest. To dissect the connection of CD271 and CD133 we have analyzed 10 patient-derived melanoma-cell strains for cell-surface expression of both markers compared to established cell lines MeWo and A375. We found CD271+ cells in the majority of cell strains analyzed as well as in a set of 16 different patient-derived melanoma metastases. Strikingly, only 2/12 cell strains harbored a CD133+ sub-set that in addition comprised a fraction of cells of a CD271+/CD133+ phenotype. Those cells were found in the label-retaining fraction and in vitro deduced from CD271+ but not CD271 knock-down cells. Conclusions: Our present study provides a deeper insight into the regulation of melanoma cell properties and points CD271 out as a regulator of several melanoma-associated genes. Further, our data strongly suggest that CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity. © 2014 Redmer et al.


Grant
Agency: European Commission | Branch: H2020 | Program: MSCA-RISE | Phase: MSCA-RISE-2016 | Award Amount: 945.00K | Year: 2017

Severe ocular disorders are affecting the lives of more than 100Mill people world-wide and at least 25% of the population above 70 years of age, a growing demographic group in EU. More than 8 million people lose their lives to cancer every year, making cancer a leading cause of pre-mature mortality in the world. The main hallmarks of severe eye conditions (i.e angiogenesis, inflammation and vascular permeability) play also pivotal roles in cancer, being therapeutic targets to treat both kind of diseases. The overall goal of 3D-NEONET is the improvement of available treatments for cancer and ocular disease by enhancing drug discovery-development and delivery to targeted tissues, through advanced international co-operation between academic and non-academic partners. The interdisciplinary expertise provided by 18 partners in 7 countries encompasses among others: drug screens, ADME, toxicology, preclinical models, nanotechnology, biomaterials and clinical trials. After the success with ongoing FP7-IAPP project 3D-NET (Drug Discovery and Development of Novel Eye Therapeutics; (www.ucd.ie/3dnet), we are assembling 3D-NEONET, this enlarged European interdisciplinary consortium that will join forces and exchange skills to enhance current therapies in oncology and ophthalmology. The 3 global objectives of 3D-NEONET are: 1- Enhance the discovery and development of novel drugs, targets and biomarkers for ophthalmology and oncology. 2- Improve the Delivery of Therapeutics for Oncology and Ophthalmology 3- Enhancement of Research, Commercial and Clinical Trial Project Management Practices in these fields. Through participation in the program, 3D-NEONET is the vehicle for driving synergies between academic and non-academic partners leading to increased scientific and technological excellence as well as tangible innovative outputs that will strengthen the competitiveness of both the researchers and industries of the network even beyond the lifetime of the network.


Karlsson J.O.G.,Linköping University | Karlsson J.O.G.,PledPharma AB | Kurz T.,Linköping University | Flechsig S.,Experimental Pharmacology and Oncology Berlin Buch GmbH | And 2 more authors.
Translational Oncology | Year: 2012

Mangafodipir is a magnetic resonance imaging contrast agent with manganese superoxide dismutase (MnSOD) mimetic activity. The MnSOD mimetic activity protects healthy cells against oxidative stress-induced detrimental effects, e.g., myelosuppressive effects of chemotherapy drugs. The contrast property depends on in vivo dissociation of Mn2+ from mangafodipir-about 80% dissociates after injection. The SOD mimetic activity, however, depends on the intact Mn complex. Complexed Mn2+ is readily excreted in the urine, whereas dissociated Mn2+ is excreted slowly via the biliary route. Mn is an essential but also a potentially neurotoxic metal. For more frequent therapeutic use, neurotoxicity due to Mn accumulation in the brain may represent a serious problem. Replacement of 4/5 of Mn2+ in mangafodipir with Ca2+ (resulting in calmangafodipir) stabilizes it from releasing Mn2+ after administration, which roughly doubles renal excretion of Mn. A considerable part of Mn2+ release from mangafodipir is governed by the presence of a limited amount of plasma zinc (Zn2+). Zn2+ has roughly 103 and 109 times higher affinity than Mn2+ and Ca2+, respectively, for fodipir. Replacement of 80%of Mn2+ with Ca2+ is enough for binding a considerable amount of the readily available plasma Zn2+, resulting in considerably less Mn2+ release and retention in the brain and other organs. At equivalent Mn2+ doses, calmangafodipir was significantly more efficacious than mangafodipir to protect BALB/c mice against myelosuppressive effects of the chemotherapy drug oxaliplatin. Calmangafodipir did not interfere negatively with the antitumor activity of oxaliplatin in CT26 tumor-bearing syngenic BALB/c mice, contrary calmangafodipir increased the antitumor activity. © 2012 Neoplasia Press, Inc. All rights reserved.


Orthmann A.,Max Delbrück Center for Molecular Medicine | Zeisig R.,Experimental Pharmacology and Oncology Berlin Buch GmbH | Suss R.,Albert Ludwigs University of Freiburg | Lorenz D.,Leibniz Institute for Molecular Pharmacology | And 2 more authors.
Pharmaceutical Research | Year: 2012

Purpose: To test targeted liposomes in an effort to improve drug transport across cellular barriers into the brain. Methods: Therefore we prepared Mitoxantrone (MTO) entrapping, rigid and fluid liposomes, equipped with a 19-mer angiopeptide as ligand for LDL lipoprotein receptor related protein (LRP) targeting. Results: Fluid, ligand bearing liposomes showed in vitro the highest cellular uptake and transcytosis and were significantly better than the corresponding ligand-free liposomes and rigid, ligand-bearing vesicles. Treatment of mice, transplanted with human breast cancer cells subcutaneously and into the brain, with fluid membrane liposomes resulted in a significant reduction in the tumor volume by more than 80% and in a clear reduction in drug toxicity. The improvement was mainly depended on liposome fluidity while the targeting contributed only to a minor degree. Pharmacokinetic parameters were also improved for liposomal MTO formulations in comparison to the free drug. So the area under the curve was increased and t 1/2 was extended for liposomes. Conclusion: Our data show that it is possible to significantly improve the therapy of brain metastases if MTO-encapsulating, fluid membrane liposomes are used instead of free MTO. This effect could be further enhanced by fluid, ligand bearing liposomes. © 2012 Springer Science+Business Media, LLC.


Cheret C.,Max Delbrück Center for Molecular Medicine | Willem M.,Ludwig Maximilians University of Munich | Fricker F.R.,University of Oxford | Wende H.,Max Delbrück Center for Molecular Medicine | And 10 more authors.
EMBO Journal | Year: 2013

The protease β-secretase 1 (Bace1) was identified through its critical role in production of amyloid-β peptides (Aβ), the major component of amyloid plaques in Alzheimer's disease. Bace1 is considered a promising target for the treatment of this pathology, but processes additional substrates, among them Neuregulin-1 (Nrg1). Our biochemical analysis indicates that Bace1 processes the Ig-containing β1 Nrg1 (IgNrg1β1) isoform. We find that a graded reduction in IgNrg1 signal strength in vivo results in increasingly severe deficits in formation and maturation of muscle spindles, a proprioceptive organ critical for muscle coordination. Further, we show that Bace1 is required for formation and maturation of the muscle spindle. Finally, pharmacological inhibition and conditional mutagenesis in adult animals demonstrate that Bace1 and Nrg1 are essential to sustain muscle spindles and to maintain motor coordination. Our results assign to Bace1 a role in the control of coordinated movement through its regulation of muscle spindle physiology, and implicate IgNrg1-dependent processing as a molecular mechanism. © 2013 European Molecular Biology Organization.


PubMed | Martin Luther University of Halle Wittenberg, Albert Ludwigs University of Freiburg, Hybrigenics, Goethe University Frankfurt and 3 more.
Type: | Journal: Oncogenesis | Year: 2015

We have previously described novel histone acetyltransferase (HAT) inhibitors that block neuroblastoma cell growth in vitro. Here we show that two selected pyridoisothiazolone HAT inhibitors, PU139 and PU141, induce cellular histone hypoacetylation and inhibit growth of several neoplastic cell lines originating from different tissues. Broader in vitro selectivity profiling shows that PU139 blocks the HATs Gcn5, p300/CBP-associated factor (PCAF), CREB (cAMP response element-binding) protein (CBP) and p300, whereas PU141 is selective toward CBP and p300. The pan-inhibitor PU139 triggers caspase-independent cell death in cell culture. Both inhibitors block growth of SK-N-SH neuroblastoma xenografts in mice and the PU139 was shown to synergize with doxorubicin in vivo. The latter also reduces histone lysine acetylation in vivo at concentrations that block neoplastic xenograft growth. This is one of the very few reports on hypoacetylating agents with in vivo anticancer activity.


Sheean M.E.,Max Delbrück Center for Molecular Medicine | McShane E.,Max Delbrück Center for Molecular Medicine | Cheret C.,Max Delbrück Center for Molecular Medicine | Walcher J.,Max Delbrück Center for Molecular Medicine | And 12 more authors.
Genes and Development | Year: 2014

Myelination depends on the synthesis of large amounts of myelin transcripts and proteins and is controlled by Nrg1/ErbB/Shp2 signaling. We developed a novel pulse labeling strategy based on stable isotope labeling with amino acids in cell culture (SILAC) to measure the dynamics of myelin protein production in mice. We found that protein synthesis is dampened in the maturing postnatal peripheral nervous system, and myelination then slows down. Remarkably, sustained activation of MAPK signaling by expression of the Mek1DD allele in mice overcomes the signals that end myelination, resulting in continuous myelin growth. MAPK activation leads to minor changes in transcript levels but massively up-regulates protein production. Pharmacological interference in vivo demonstrates that the effects of activated MAPK signaling on translation are mediated by mTORindependent mechanisms but in part also by mTOR-dependent mechanisms. Previous work demonstrated that loss of ErbB3/Shp2 signaling impairs Schwann cell development and disrupts the myelination program. We found that activated MAPK signaling strikingly compensates for the absence of ErbB3 or Shp2 during Schwann cell development and myelination. © 2014 Sheean et al.; Published by Cold Spring Harbor Laboratory Press.


PubMed | McGowan Institute for Regenerative Medicine, Experimental Pharmacology and Oncology Berlin Buch GmbH and Charité - Medical University of Berlin
Type: Journal Article | Journal: Biotechnology progress | Year: 2016

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale-up of stem cell culture is necessary. Bioreactors for dynamic three-dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow-fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 10(6) mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 10(6) mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four-compartment bioreactor including further cultivation of remaining cells.


Schallschmidt K.,BAM Federal Institute of Materials Research and Testing | Becker R.,BAM Federal Institute of Materials Research and Testing | Zwaka H.,Free University of Berlin | Menzel R.,Free University of Berlin | And 4 more authors.
Journal of Breath Research | Year: 2015

In vitro cultured lung cancer cell lines were investigated regarding the possible identification of volatile organic compounds as potential biomarkers. Gas samples from the headspace of pure culture medium and from the cultures of human lung adenocarcinoma cell lines A549 and Lu7466 were exposed to polypropylene fleece in order to absorb odour components. Sniffer dogs were trained with loaded fleeces of both cell lines, and honey bees were trained with fleeces exposed to A549. Afterwards, their ability to distinguish between cell-free culture medium odour and lung cancer cell odour was tested. Neither bees nor dogs were able to discriminate between odours from the cancer cell cultures and the pure culture medium. Solid phase micro extraction followed by gas chromatography with mass selective detection produced profiles of volatiles from the headspace offered to the animals. The profiles from the cell lines were largely similar; distinct differences were based on the decrease of volatile culture medium components due to the cells' metabolic activity. In summary, cultured lung cancer cell lines do not produce any biomarkers recognizable by animals or gas chromatographic analysis. © 2015 IOP Publishing Ltd.


PubMed | Experimental Pharmacology and Oncology Berlin Buch GmbH, University of Wisconsin - Madison and City of Hope Comprehensive Cancer Center
Type: Journal Article | Journal: Science signaling | Year: 2017

The epidermal growth factor receptor (EGFR) is a therapeutic target in patients with various cancers. Unfortunately, resistance to EGFR-targeted therapeutics is common. Previous studies identified two mechanisms of resistance to the EGFR monoclonal antibody cetuximab. Nuclear translocation of EGFR bypasses the inhibitory effects of cetuximab, and the receptor tyrosine kinase AXL mediates cetuximab resistance by maintaining EGFR activation and downstream signaling. Thus, we hypothesized that AXL mediated the nuclear translocation of EGFR in the setting of cetuximab resistance. Cetuximab-resistant clones of non-small cell lung cancer in culture and patient-derived xenografts in mice had increased abundance of AXL and nuclear EGFR (nEGFR). Cellular fractionation analysis, super-resolution microscopy, and electron microscopy revealed that genetic loss of AXL reduced the accumulation of nEGFR. SRC family kinases (SFKs) and HER family ligands promote the nuclear translocation of EGFR. We found that AXL knockdown reduced the expression of the genes encoding the SFK family members YES and LYN and the ligand neuregulin-1 (NRG1). AXL knockdown also decreased the interaction between EGFR and the related receptor HER3 and accumulation of HER3 in the nucleus. Overexpression of LYN and NRG1 in cells depleted of AXL resulted in accumulation of nEGFR, rescuing the deficit induced by lack of AXL. Collectively, these data uncover a previously unrecognized role for AXL in regulating the nuclear translocation of EGFR and suggest that AXL-mediated SFK and NRG1 expression promote this process.

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