Publications by authors named "Thorsten Stiewe"

96 Publications

Efficient antisense inhibition reveals microRNA-155 to restrain a late-myeloid inflammatory programme in primary human phagocytes.

RNA Biol 2021 Feb 23:1-15. Epub 2021 Feb 23.

Department of Medicine, Institute for Lung Research, Philipps University Marburg, Marburg, Germany.

A persisting obstacle in human immunology is that blood-derived leukocytes are notoriously difficult to manipulate at the RNA level. Therefore, our knowledge about immune-regulatory RNA-networks is largely based on tumour cell-line and rodent knockout models, which do not fully mimic human leukocyte biology. Here, we exploit straightforward cell penetrating peptide (CPP) chemistry to enable efficient loss-of-function phenotyping of regulatory RNAs in primary human blood-derived cells. The classical CPP octaarginine (R8) enabled antisense peptide-nucleic-acid (PNA) oligomer delivery into nearly 100% of human blood-derived macrophages without apparent cytotoxicity even up to micromolar concentrations. In a proof-of-principle experiment, we successfully de-repressed the global microRNA-155 regulome in primary human macrophages using a PNA-R8 oligomer, which phenocopies a CRISPR-Cas9 induced gene knockout. Interestingly, although it is often believed that fairly high concentrations (μM) are needed to achieve antisense activity, our PNA-R8 was effective at 200 nM. RNA-seq characterized microRNA-155 as a broad-acting riboregulator, feedback restraining a late myeloid differentiation-induced pro-inflammatory network, comprising MyD88-signalling and ubiquitin-proteasome components. Our results highlight the important role of the microRNA machinery in fine-control of blood-derived human phagocyte immunity and open the door for further studies on regulatory RNAs in difficult-to-transfect primary human immune cells.
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http://dx.doi.org/10.1080/15476286.2021.1885209DOI Listing
February 2021

Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis.

PLoS Genet 2021 Feb 18;17(2):e1009318. Epub 2021 Feb 18.

Institute of Molecular Biology and Tumor Research, Biomedical Research Center, Philipps-University, Marburg, Germany.

The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.
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http://dx.doi.org/10.1371/journal.pgen.1009318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891773PMC
February 2021

C1q/TNF-Related Protein 3 (CTRP-3) Deficiency of Adipocytes Affects White Adipose Tissue Mass but Not Systemic CTRP-3 Concentrations.

Int J Mol Sci 2021 Feb 7;22(4). Epub 2021 Feb 7.

Department of Internal Medicine III, University of Giessen, 35390 Giessen, Germany.

CTRP-3 (C1q/TNF-related protein-3) is an adipokine with endocrine and immunological function. The impact of adipocyte CTRP-3 production on systemic CTRP-3 concentrations and on adipocyte biology is unknown. A murine model of adipocyte CTRP-3 knockout (KO) was established (via the system). Serum adipokine levels were quantified by ELISA and adipose tissue (AT) gene expression by real-time PCR. Preadipocytes were isolated from AT and differentiated into adipocytes. Comparative transcriptome analysis was applied in adipocytes and liver tissue. Body weight and AT mass were reduced in CTRP-3 KO mice together with decreased serum leptin. In primary cells from visceral AT of KO mice, expression of adiponectin, progranulin, and resistin was induced, while peroxisome proliferator activated receptor γ () was decreased. M1/M2 macrophage polarization markers were shifted to a more anti-inflammatory phenotype. CTRP-3 expression in AT did not contribute to serum concentrations. AT and liver morphology remained unaffected by CTRP-3 KO. Myelin transcription factor 1-like () was identified as a highly upregulated gene. In conclusion, adipocyte CTRP-3 has a role in adipogenesis and AT weight gain whereas adipocyte differentiation is not impaired by CTRP-3 deficiency. Since no effects on circulating CTRP-3 levels were observed, the impact of adipocyte CTRP-3 KO is limited to adipose tissue. Modified AT gene expression indicates a rather anti-inflammatory phenotype.
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http://dx.doi.org/10.3390/ijms22041670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915696PMC
February 2021

JAZF1, A Novel p400/TIP60/NuA4 Complex Member, Regulates H2A.Z Acetylation at Regulatory Regions.

Int J Mol Sci 2021 Jan 12;22(2). Epub 2021 Jan 12.

Institute for Genetics, Justus-Liebig University Giessen, 35392 Giessen, Germany.

Histone variants differ in amino acid sequence, expression timing and genomic localization sites from canonical histones and convey unique functions to eukaryotic cells. Their tightly controlled spatial and temporal deposition into specific chromatin regions is accomplished by dedicated chaperone and/or remodeling complexes. While quantitatively identifying the chaperone complexes of many human H2A variants by using mass spectrometry, we also found additional members of the known H2A.Z chaperone complexes p400/TIP60/NuA4 and SRCAP. We discovered JAZF1, a nuclear/nucleolar protein, as a member of a p400 sub-complex containing MBTD1 but excluding ANP32E. Depletion of JAZF1 results in transcriptome changes that affect, among other pathways, ribosome biogenesis. To identify the underlying molecular mechanism contributing to JAZF1's function in gene regulation, we performed genome-wide ChIP-seq analyses. Interestingly, depletion of JAZF1 leads to reduced H2A.Z acetylation levels at > 1000 regulatory sites without affecting H2A.Z nucleosome positioning. Since JAZF1 associates with the histone acetyltransferase TIP60, whose depletion causes a correlated H2A.Z deacetylation of several JAZF1-targeted enhancer regions, we speculate that JAZF1 acts as chromatin modulator by recruiting TIP60's enzymatic activity. Altogether, this study uncovers JAZF1 as a member of a TIP60-containing p400 chaperone complex orchestrating H2A.Z acetylation at regulatory regions controlling the expression of genes, many of which are involved in ribosome biogenesis.
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http://dx.doi.org/10.3390/ijms22020678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826843PMC
January 2021

Phosphoproteomics identify arachidonic-acid-regulated signal transduction pathways modulating macrophage functions with implications for ovarian cancer.

Theranostics 2021 1;11(3):1377-1395. Epub 2021 Jan 1.

Tranlational Oncology Group, Center for Tumor Biology and Immunology, Philipps University, Marburg, Germany.

Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions. AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release. We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca-triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options. Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.
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http://dx.doi.org/10.7150/thno.52442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738879PMC
January 2021

Extracellular Vesicles: Messengers of p53 in Tumor-Stroma Communication and Cancer Metastasis.

Int J Mol Sci 2020 Dec 17;21(24). Epub 2020 Dec 17.

Institute of Molecular Oncology, Philipps University, 35034 Marburg, Germany.

Tumor progression to a metastatic and ultimately lethal stage relies on a tumor-supporting microenvironment that is generated by reciprocal communication between tumor and stromal host cells. The tumor-stroma crosstalk is instructed by the genetic alterations of the tumor cells-the most frequent being mutations in the gene () that are clinically correlated with metastasis, drug resistance and poor patient survival. The crucial mediators of tumor-stroma communication are tumor-derived extracellular vesicles (EVs), in particular exosomes, which operate both locally within the primary tumor and in distant organs, at pre-metastatic niches as the future sites of metastasis. Here, we review how wild-type and mutant p53 proteins control the secretion, size, and especially the RNA and protein cargo of tumor-derived EVs. We highlight how EVs extend the cell-autonomous tumor suppressive activity of wild-type p53 into the tumor microenvironment (TME), and how mutant p53 proteins switch EVs into oncogenic messengers that reprogram tumor-host communication within the entire organism so as to promote metastatic tumor cell dissemination.
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http://dx.doi.org/10.3390/ijms21249648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766631PMC
December 2020

Epigenetic Inactivation of the Tumor Suppressor Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis.

Cancers (Basel) 2020 Nov 26;12(12). Epub 2020 Nov 26.

Faculty of Biology, Institute for Genetics, Justus-Liebig-University Giessen, 35392 Giessen, Germany.

(IRX) encodes members of homeodomain containing genes which are involved in development and differentiation. Since it has been reported that the gene is localized in a lung cancer susceptibility locus, the epigenetic regulation and function of IRX1 was investigated in lung carcinogenesis. We observed frequent hypermethylation of the promoter in non-small cell lung cancer (NSCLC) compared to small cell lung cancer (SCLC). Aberrant methylation was significantly correlated with reduced expression. In normal lung samples, the promoter showed lower median DNA methylation levels (<10%) compared to primary adenocarcinoma (ADC, 22%) and squamous cell carcinoma (SQCC, 14%). A significant hypermethylation and downregulation of was detected in ADC and SQCC compared to matching normal lung samples ( < 0.0001). Low expression was significantly correlated with impaired prognosis of ADC patients ( = 0.001). Reduced survival probability was also associated with higher promoter methylation ( = 0.02). Inhibition of DNA methyltransferase (DNMT) activity reactivated expression in human lung cancer cell lines. Induced DNMT3A and EZH2 expression was correlated with downregulation of . On the cellular level, IRX1 exhibits nuclear localization and expression of IRX1 induced fragmented nuclei in cancer cells. Localization of IRX1 and induction of aberrant nuclei were dependent on the presence of the homeobox of IRX1. By data mining, we showed that is negatively correlated with oncogenic pathways and expression induces the proapoptotic regulator . In conclusion, we report that expression is significantly associated with improved survival probability of ADC patients. hypermethylation may serve as molecular biomarker for ADC diagnosis and prognosis. Our data suggest that acts as an epigenetically regulated tumor suppressor in the pathogenesis of lung cancer.
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http://dx.doi.org/10.3390/cancers12123528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760495PMC
November 2020

Modulation of pancreatic neuroendocrine neoplastic cell fate by autophagy mediated death.

Neuroendocrinology 2020 Oct 27. Epub 2020 Oct 27.

Introduction Autophagic cell death in cancer cells can be mediated by inhibition of deacetylases. Although extensive studies have focused on the autophagic process in cancer, little is known about the role of autophagy in degrading cytosolic and nuclear components of pancreatic neuroendocrine neoplastic (pNEN) cells leading to cell death, thus improving the therapy of patients affected by pNEN. Methods 2D and 3D human pNEN and pancreatic stellate cells were treated with panobinostat and bafilomycin. Autophagy markers were detected by RT-qPCR, immunofluorescence and western blot. Autophagosomes were detected by electron microscopy and their maturation by real time fluorescence of LC3B stable transfected cells. ChIP was performed at the cAMP responsive element. Immunofluorescence was performed in murine pancreatic tissue. Results We observed that pan-deacetylase inhibitor panobinostat treatment causes autophagic cell death in pNEN cells. We also found that although AMPK-α phosphorylation is counterbalanced by phosphorylated AKT, it is not capable to inhibiting autophagic cell death. Whereas the binding activity of cAMP responsive element is prompted by panobinostat. Although autophagy inhibition prevented autophagosomes synthesis, maturation and cell death, panobinostat treatment induced the accumulation of mature autophagosomes in the cytosol, and the nucleus, leading to disruption of the organelles, cellular digestion and decay. Observation of autophagosome membrane proteins Beclin1 and LC3B aggregation in murine pancreatic islets indicates that autophagy restoration may also lead to autophagosome aggregation in murine insulinoma cells. A basal low expression of autophagy markers was detectable in patients affected by pNEN and, interestingly, the expression of these markers was significantly lower in metastatic pNEN. Discussion/Conclusion Our study highlights that the autophagy functional restoration and prolongation of this catabolic process, mediated by inhibition of deacetylase, is responsible for the reduction of pNEN cells. Prompting of autophagy cell death could be a promising strategy for the therapy of pNEN.
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http://dx.doi.org/10.1159/000512567DOI Listing
October 2020

mTOR-mediated cancer drug resistance suppresses autophagy and generates a druggable metabolic vulnerability.

Nat Commun 2020 09 17;11(1):4684. Epub 2020 Sep 17.

Institute of Molecular Oncology, Philipps-University, Marburg, Germany.

Cancer cells have a characteristic metabolism, mostly caused by alterations in signal transduction networks rather than mutations in metabolic enzymes. For metabolic drugs to be cancer-selective, signaling alterations need to be identified that confer a druggable vulnerability. Here, we demonstrate that many tumor cells with an acquired cancer drug resistance exhibit increased sensitivity to mechanistically distinct inhibitors of cancer metabolism. We demonstrate that this metabolic vulnerability is driven by mTORC1, which promotes resistance to chemotherapy and targeted cancer drugs, but simultaneously suppresses autophagy. We show that autophagy is essential for tumor cells to cope with therapeutic perturbation of metabolism and that mTORC1-mediated suppression of autophagy is required and sufficient for generating a metabolic vulnerability leading to energy crisis and apoptosis. Our study links mTOR-induced cancer drug resistance to autophagy defects as a cause of a metabolic liability and opens a therapeutic window for the treatment of otherwise therapy-refractory tumor patients.
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http://dx.doi.org/10.1038/s41467-020-18504-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499183PMC
September 2020

Phosphorylation Control of p53 DNA-Binding Cooperativity Balances Tumorigenesis and Aging.

Cancer Res 2020 12 1;80(23):5231-5244. Epub 2020 Sep 1.

Institute of Molecular Oncology, Member of the German Center for Lung Research (DZL), Philipps-University Marburg, Marburg, Germany.

Posttranslational modifications are essential for regulating the transcription factor p53, which binds DNA in a highly cooperative manner to control expression of a plethora of tumor-suppressive programs. Here we show at the biochemical, cellular, and organismal level that the cooperative nature of DNA binding is reduced by phosphorylation of highly conserved serine residues (human S183/S185, mouse S180) in the DNA-binding domain. To explore the role of this inhibitory phosphorylation , new phosphorylation-deficient p53-S180A knock-in mice were generated. Chromatin immunoprecipitation sequencing and RNA sequencing studies of S180A knock-in cells demonstrated enhanced DNA binding and increased target gene expression. , this translated into a tissue-specific vulnerability of the bone marrow that caused depletion of hematopoietic stem cells and impaired proper regeneration of hematopoiesis after DNA damage. Median lifespan was significantly reduced by 20% from 709 days in wild type to only 568 days in S180A littermates. Importantly, lifespan was reduced by a loss of general fitness and increased susceptibility to age-related diseases, not by increased cancer incidence as often seen in other p53-mutant mouse models. For example, S180A knock-in mice showed markedly reduced spontaneous tumorigenesis and increased resistance to -driven lymphoma and -driven lung cancer. Preventing phosphorylation of S183/S185 in human cells boosted p53 activity and allowed tumor cells to be killed more efficiently. Together, our data identify p53 DNA-binding domain phosphorylation as a druggable mechanism that balances tumorigenesis and aging. SIGNIFICANCE: These findings demonstrate that p53 tumor suppressor activity is reduced by DNA-binding domain phosphorylation to prevent aging and identify this phosphorylation as a potential target for cancer therapy..
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2002DOI Listing
December 2020

Metastasis-Associated Protein 2 Represses NF-κB to Reduce Lung Tumor Growth and Inflammation.

Cancer Res 2020 10 14;80(19):4199-4211. Epub 2020 Aug 14.

Max Planck Institute for Heart and Lung Research, German Center for Lung Research (DZL), Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany.

Although NF-κB is known to play a pivotal role in lung cancer, contributing to tumor growth, microenvironmental changes, and metastasis, the epigenetic regulation of NF-κB in tumor context is largely unknown. Here we report that the IKK2/NF-κB signaling pathway modulates metastasis-associated protein 2 (MTA2), a component of the nucleosome remodeling and deacetylase complex (NuRD). In triple transgenic mice, downregulation of IKK2 (Sftpc-cRaf-IKK2DN) in cRaf-induced tumors in alveolar epithelial type II cells restricted tumor formation, whereas activation of IKK2 (Sftpc-cRaf-IKK2CA) supported tumor growth; both effects were accompanied by altered expression of MTA2. Further studies employing genetic inhibition of MTA2 suggested that in primary tumor growth, independent of IKK2, MTA2/NuRD corepressor complex negatively regulates NF-κB signaling and tumor growth, whereas later dissociation of MTA2/NuRD complex from the promoter of NF-κB target genes and IKK2-dependent positive regulation of MTA2 leads to activation of NF-κB signaling, epithelial-mesenchymal transition, and lung tumor metastasis. These findings reveal a previously unrecognized biphasic role of MTA2 in IKK2/NF-κB-driven primary-to-metastatic lung tumor progression. Addressing the interaction between MTA2 and NF-κB would provide potential targets for intervention of tumor growth and metastasis. SIGNIFICANCE: These findings strongly suggest a prominent role of MTA2 in primary tumor growth, lung metastasis, and NF-κB signaling modulatory functions.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1158DOI Listing
October 2020

The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic.

Nature 2020 10 29;586(7827):133-138. Epub 2020 Jul 29.

The Lautenberg Center for Immunology and Cancer Research, Institute of Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Somatic mutations in p53, which inactivate the tumour-suppressor function of p53 and often confer oncogenic gain-of-function properties, are very common in cancer. Here we studied the effects of hotspot gain-of-function mutations in Trp53 (the gene that encodes p53 in mice) in mouse models of WNT-driven intestinal cancer caused by Csnk1a1 deletion or Apc mutation. Cancer in these models is known to be facilitated by loss of p53. We found that mutant versions of p53 had contrasting effects in different segments of the gut: in the distal gut, mutant p53 had the expected oncogenic effect; however, in the proximal gut and in tumour organoids it had a pronounced tumour-suppressive effect. In the tumour-suppressive mode, mutant p53 eliminated dysplasia and tumorigenesis in Csnk1a1-deficient and Apc mice, and promoted normal growth and differentiation of tumour organoids derived from these mice. In these settings, mutant p53 was more effective than wild-type p53 at inhibiting tumour formation. Mechanistically, the tumour-suppressive effects of mutant p53 were driven by disruption of the WNT pathway, through preventing the binding of TCF4 to chromatin. Notably, this tumour-suppressive effect was completely abolished by the gut microbiome. Moreover, a single metabolite derived from the gut microbiota-gallic acid-could reproduce the entire effect of the microbiome. Supplementing gut-sterilized p53-mutant mice and p53-mutant organoids with gallic acid reinstated the TCF4-chromatin interaction and the hyperactivation of WNT, thus conferring a malignant phenotype to the organoids and throughout the gut. Our study demonstrates the substantial plasticity of a cancer mutation and highlights the role of the microenvironment in determining its functional outcome.
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http://dx.doi.org/10.1038/s41586-020-2541-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116712PMC
October 2020

Cyclophilin inhibitors restrict Middle East respiratory syndrome coronavirus interferon-λ and in mice.

Eur Respir J 2020 Nov 26;56(5). Epub 2020 Nov 26.

Dept of Internal Medicine II, University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany.

While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV), cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use.We elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin A (CsA) and alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily available therapy in MERS-CoV infection.Calu-3 cells and primary human alveolar epithelial cells (hAECs) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nuclear factor of activated T-cells (NFATs) or mitogen-activated protein kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by quantitative real-time PCR and 50% tissue culture infective dose. Data were validated in a murine MERS-CoV infection model.Both CsA and ALV reduced MERS-CoV titres and viral RNA replication in Calu-3 cells and hAECs, improving epithelial integrity. While neither calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type III interferon (IFNλ) response and expression of antiviral genes. Downregulation of IRF1 or IFNλ increased MERS-CoV propagation in the presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication , correlating with elevated lung IFNλ levels and improved outcome.We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication and upregulation of inflammatory antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate for treating MERS-CoV infection.
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http://dx.doi.org/10.1183/13993003.01826-2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331652PMC
November 2020

Reprogramming of tumor-associated macrophages by targeting β-catenin/FOSL2/ARID5A signaling: A potential treatment of lung cancer.

Sci Adv 2020 Jun 5;6(23):eaaz6105. Epub 2020 Jun 5.

Max Planck Institute for Heart and Lung Research, Member of the German Center for Lung Research (DZL), Member of the Cardio-Pulmonary Institute (CPI), Bad Nauheim 61231, Germany.

Tumor-associated macrophages (TAMs) influence lung tumor development by inducing immunosuppression. Transcriptome analysis of TAMs isolated from human lung tumor tissues revealed an up-regulation of the Wnt/β-catenin pathway. These findings were reproduced in a newly developed in vitro "trained" TAM model. Pharmacological and macrophage-specific genetic ablation of β-catenin reprogrammed M2-like TAMs to M1-like TAMs both in vitro and in various in vivo models, which was linked with the suppression of primary and metastatic lung tumor growth. An in-depth analysis of the underlying signaling events revealed that β-catenin-mediated transcriptional activation of FOS-like antigen 2 (FOSL2) and repression of the AT-rich interaction domain 5A (ARID5A) drive gene regulatory switch from M1-like TAMs to M2-like TAMs. Moreover, we found that high expressions of β-catenin and FOSL2 correlated with poor prognosis in patients with lung cancer. In conclusion, β-catenin drives a transcriptional switch in the lung tumor microenvironment, thereby promoting tumor progression and metastasis.
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http://dx.doi.org/10.1126/sciadv.aaz6105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274802PMC
June 2020

Upregulation of mesothelial genes in ovarian carcinoma cells is associated with an unfavorable clinical outcome and the promotion of cancer cell adhesion.

Mol Oncol 2020 09 25;14(9):2142-2162. Epub 2020 Jun 25.

Center for Tumor Biology and Immunology, Philipps University, Marburg, Germany.

A hallmark of ovarian high-grade serous carcinoma (HGSC) is its early and massive peritoneal dissemination via the peritoneal fluid. It is generally believed that tumor cells must breach the mesothelium of peritoneal organs to adhere to the underlying extracellular matrix (ECM) and initiate metastatic growth. However, the molecular mechanisms underlying these processes are only partially understood. Here, we have analyzed 52 matched samples of spheroids and solid tumor masses (suspected primary lesions and metastases) from 10 patients by targeted sequencing of 21 loci previously proposed as targets of HGSC driver mutations. This analysis revealed very similar patterns of genetic alterations in all samples. One exception was FAT3 with a strong enrichment of mutations in metastases compared with presumed primary lesions in two cases. FAT3 is a putative tumor suppressor gene that codes for an atypical cadherin, pointing a potential role in peritoneal dissemination in a subgroup of HGSC patients. By contrast, transcriptome data revealed clear and consistent differences between tumor cell spheroids from ascites and metastatic lesions, which were mirrored by the in vitro adherence of ascites-derived spheroids. The adhesion-induced transcriptional alterations in metastases and adherent cells resembled epithelial-mesenchymal transition, but surprisingly also included the upregulation of a specific subset of mesothelial genes, such as calretinin (CALB2) and podoplanin (PDPN). Consistent with this finding, calretinin staining was also observed in subsets of tumor cells in HGSC metastases, particularly at the invasive tumor edges. Intriguingly, a high expression of either CALB2 or PDPN was strongly associated with a poor clinical outcome. siRNA-mediated CALB2 silencing triggered the detachment of adherent HGSC cells in vitro and inhibited the adhesion of detached HGSC cells to collagen type I. Our data suggest that the acquisition of a mesenchymal-mesothelial phenotype contributes to cancer cell adhesion to the ECM of peritoneal organs and HGSC progression.
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http://dx.doi.org/10.1002/1878-0261.12749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463315PMC
September 2020

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution.

Nat Commun 2020 05 13;11(1):2381. Epub 2020 May 13.

Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 10, 35043, Marburg, Germany.

Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.
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http://dx.doi.org/10.1038/s41467-020-16169-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221075PMC
May 2020

YM155-Adapted Cancer Cell Lines Reveal Drug-Induced Heterogeneity and Enable the Identification of Biomarker Candidates for the Acquired Resistance Setting.

Cancers (Basel) 2020 Apr 26;12(5). Epub 2020 Apr 26.

Institute for Medical Virology, University Hospital, Goethe University Frankfurt am Main, 60596 Frankfurt, Germany.

Survivin is a drug target and its suppressant YM155 a drug candidate mainly investigated for high-risk neuroblastoma. Findings from one YM155-adapted subline of the neuroblastoma cell line UKF-NB-3 had suggested that increased ABCB1 (mediates YM155 efflux) levels, decreased SLC35F2 (mediates YM155 uptake) levels, decreased survivin levels, and mutations indicate YM155 resistance. Here, the investigation of 10 additional YM155-adapted UKF-NB-3 sublines only confirmed the roles of ABCB1 and SLC35F2. However, cellular ABCB1 and SLC35F2 levels did not indicate YM155 sensitivity in YM155-naïve cells, as indicated by drug response data derived from the Cancer Therapeutics Response Portal (CTRP) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases. Moreover, the resistant sublines were characterized by a remarkable heterogeneity. Only seven sublines developed on-target resistance as indicated by resistance to RNAi-mediated survivin depletion. The sublines also varied in their response to other anti-cancer drugs. In conclusion, cancer cell populations of limited intrinsic heterogeneity can develop various resistance phenotypes in response to treatment. Therefore, individualized therapies will require monitoring of cancer cell evolution in response to treatment. Moreover, biomarkers can indicate resistance formation in the acquired resistance setting, even when they are not predictive in the intrinsic resistance setting.
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http://dx.doi.org/10.3390/cancers12051080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281096PMC
April 2020

Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways.

J Biol Chem 2020 08 17;295(33):11388-11407. Epub 2020 Apr 17.

Institute of Virology, Philipps-University, 35043 Marburg, Germany

Cleavage of influenza virus hemagglutinin (HA) by host proteases is essential for virus infectivity. HA of most influenza A and B (IAV/IBV) viruses is cleaved at a monobasic motif by trypsin-like proteases. Previous studies have reported that transmembrane serine protease 2 (TMPRSS2) is essential for activation of H7N9 and H1N1pdm IAV in mice but that H3N2 IAV and IBV activation is independent of TMPRSS2 and carried out by as-yet-undetermined protease(s). Here, to identify additional H3 IAV- and IBV-activating proteases, we used RNA-Seq to investigate the protease repertoire of murine lower airway tissues, primary type II alveolar epithelial cells (AECIIs), and the mouse lung cell line MLE-15. Among 13 candidates identified, TMPRSS4, TMPRSS13, hepsin, and prostasin activated H3 and IBV HA IBV activation and replication was reduced in AECIIs from -deficient mice compared with WT or Tmprss2-deficient mice, indicating that murine TMPRSS4 is involved in IBV activation. Multicycle replication of H3N2 IAV and IBV in AECIIs of -deficient mice varied in sensitivity to protease inhibitors, indicating that different, but overlapping, sets of murine proteases facilitate H3 and IBV HA cleavages. Interestingly, human hepsin and prostasin orthologs did not activate H3, but they did activate IBV HA Our results indicate that TMPRSS4 is an IBV-activating protease in murine AECIIs and suggest that TMPRSS13, hepsin, and prostasin cleave H3 and IBV HA in mice. They further show that hepsin and prostasin orthologs might contribute to the differences observed in TMPRSS2-independent activation of H3 in murine and human airways.
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http://dx.doi.org/10.1074/jbc.RA120.012635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450141PMC
August 2020

HDAC3 functions as a positive regulator in Notch signal transduction.

Nucleic Acids Res 2020 04;48(7):3496-3512

Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany.

Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.
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http://dx.doi.org/10.1093/nar/gkaa088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144913PMC
April 2020

p53's Extended Reach: The Mutant p53 Secretome.

Biomolecules 2020 02 15;10(2). Epub 2020 Feb 15.

Institute of Molecular Oncology, Member of the German Center for Lung Research (DZL), Philipps-University, Hans-Meerwein-Straße 3, 35043 Marburg, Germany.

p53 suppresses tumorigenesis by activating a plethora of effector pathways. While most of these operate primarily inside of cells to limit proliferation and survival of incipient cancer cells, many extend to the extracellular space. In particular, p53 controls expression and secretion of numerous extracellular factors that are either soluble or contained within extracellular vesicles such as exosomes. As part of the cellular secretome, they execute key roles in cell-cell communication and extracellular matrix remodeling. Mutations in the p53-encoding gene are the most frequent genetic alterations in cancer cells, and therefore, have profound impact on the composition of the tumor cell secretome. In this review, we discuss how the loss or dominant-negative inhibition of wild-type p53 in concert with a gain of neomorphic properties observed for many mutant p53 proteins, shapes a tumor cell secretome that creates a supportive microenvironment at the primary tumor site and primes niches in distant organs for future metastatic colonization.
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http://dx.doi.org/10.3390/biom10020307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072272PMC
February 2020

Inactivation of Mdm2 restores apoptosis proficiency of cooperativity mutant p53 in vivo.

Cell Cycle 2020 01 21;19(1):109-123. Epub 2019 Nov 21.

Institute of Molecular Oncology, Member of the German Center for Lung Research (DZL), Philipps-University, Marburg, Germany.

mutations are found in 50% of all cancers and mutated status is considered poor for treatment. However, some mutations exhibit only partial loss-of-function (LOF), meaning they retain residual transcriptional and non-transcriptional activities that are potentially beneficial for therapy. Earlier we have characterized a knock-in mouse model for the partial LOF mutant (p53RR). Reduced DNA binding cooperativity of this mutant led to the loss of p53-dependent apoptosis, while p53 functions in cell cycle control, senescence, metabolism, and antioxidant defense remained intact. Concomitantly, tumor suppression was evident but strongly compromised compared to wild-type mice. Here we used the mouse as a model to investigate whether residual functions of mutant p53 can be engaged to induce cell death, which is considered the most desirable outcome of tumor therapy. We made use of knock-out in developing embryos as a sensitive tool for detecting remaining p53 activities. Genetic ablation of led to embryonic lethality in homozygotes at days 9.5-11.5. This effect was not rescued by concomitant p21-knockout, indicating its independence of p21-mediated cell cycle arrest. Instead, immunohistochemical analysis showed widespread apoptosis in tissues of defective embryos accompanied by persistent accumulation of p53RR protein. This led to partial restoration of the mutant's proficiency in transcriptional induction of the pro-apoptotic genes (Puma) and . These data indicate that increased quantity can compensate for qualitative defects of p53 mutants and suggest that Mdm2-targeting (potentially in combination with other drugs) might be effective against cells bearing p53 partial LOF mutants.
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http://dx.doi.org/10.1080/15384101.2019.1693748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927723PMC
January 2020

Distinct IL-1α-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner.

EMBO J 2020 01 7;39(1):e101533. Epub 2019 Nov 7.

Rudolf Buchheim Institute of Pharmacology, Justus Liebig University Giessen, Giessen, Germany.

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 "master" enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB.
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http://dx.doi.org/10.15252/embj.2019101533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939198PMC
January 2020

Distinct CoREST complexes act in a cell-type-specific manner.

Nucleic Acids Res 2019 12;47(22):11649-11666

Institute of Molecular Biology and Tumor Research, Biomedical Research Center, Philipps-University, Hans-Meerwein-Strasse 2, 35043, Marburg, Germany.

CoREST has been identified as a subunit of several protein complexes that generate transcriptionally repressive chromatin structures during development. However, a comprehensive analysis of the CoREST interactome has not been carried out. We use proteomic approaches to define the interactomes of two dCoREST isoforms, dCoREST-L and dCoREST-M, in Drosophila. We identify three distinct histone deacetylase complexes built around a common dCoREST/dRPD3 core: A dLSD1/dCoREST complex, the LINT complex and a dG9a/dCoREST complex. The latter two complexes can incorporate both dCoREST isoforms. By contrast, the dLSD1/dCoREST complex exclusively assembles with the dCoREST-L isoform. Genome-wide studies show that the three dCoREST complexes associate with chromatin predominantly at promoters. Transcriptome analyses in S2 cells and testes reveal that different cell lineages utilize distinct dCoREST complexes to maintain cell-type-specific gene expression programmes: In macrophage-like S2 cells, LINT represses germ line-related genes whereas other dCoREST complexes are largely dispensable. By contrast, in testes, the dLSD1/dCoREST complex prevents transcription of germ line-inappropriate genes and is essential for spermatogenesis and fertility, whereas depletion of other dCoREST complexes has no effect. Our study uncovers three distinct dCoREST complexes that function in a lineage-restricted fashion to repress specific sets of genes thereby maintaining cell-type-specific gene expression programmes.
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http://dx.doi.org/10.1093/nar/gkz1050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145674PMC
December 2019

Loss of p53 function at late stages of tumorigenesis confers ARF-dependent vulnerability to p53 reactivation therapy.

Proc Natl Acad Sci U S A 2019 10 14;116(44):22288-22293. Epub 2019 Oct 14.

Institute of Molecular Oncology, Member of the German Center for Lung Research, Philipps University Marburg, 35043 Marburg, Germany;

Cancer development is driven by activated oncogenes and loss of tumor suppressors. While oncogene inhibitors have entered routine clinical practice, tumor suppressor reactivation therapy remains to be established. For the most frequently inactivated tumor suppressor p53, genetic mouse models have demonstrated regression of p53-null tumors upon p53 reactivation. While this was shown in tumor models driven by p53 loss as the initiating lesion, many human tumors initially develop in the presence of wild-type p53, acquire aberrations in the p53 pathway to bypass p53-mediated tumor suppression, and inactivate p53 itself only at later stages during metastatic progression or therapy. To explore the efficacy of p53 reactivation in this scenario, we used a reversibly switchable p53 (p53ER) mouse allele to generate Eµ-Myc-driven lymphomas in the presence of active p53 and, after full lymphoma establishment, switched off p53 to model late-stage p53 inactivation. Although these lymphomas had evolved in the presence of active p53, later loss and subsequent p53 reactivation surprisingly activated p53 target genes triggering massive apoptosis, tumor regression, and long-term cure of the majority of animals. Mechanistically, the reactivation response was dependent on Cdkn2a/p19Arf, which is commonly silenced in p53 wild-type lymphomas, but became reexpressed upon late-stage p53 inactivation. Likewise, human p53 wild-type tumor cells with CRISPR-engineered switchable p53ER alleles responded to p53 reactivation when CDKN2A/p14ARF function was restored or mimicked with Mdm2 inhibitors. Together, these experiments provide genetic proof of concept that tumors can respond, in an ARF-dependent manner, to p53 reactivation even if p53 inactivation has occurred late during tumor evolution.
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http://dx.doi.org/10.1073/pnas.1910255116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825290PMC
October 2019

Exosome-dependent immune surveillance at the metastatic niche requires BAG6 and CBP/p300-dependent acetylation of p53.

Theranostics 2019 14;9(21):6047-6062. Epub 2019 Aug 14.

Experimental Tumor Research, Center for Tumor Biology and Immunology, Clinic for Hematology, Oncology and Immunology, Philipps University, Marburg, Germany.

Extracellular vesicles released by tumor cells contribute to the reprogramming of the tumor microenvironment and interfere with hallmarks of cancer including metastasis. Notably, melanoma cell-derived EVs are able to establish a pre-metastatic niche in distant organs, or on the contrary, exert anti-tumor activity. However, molecular insights into how vesicles are selectively packaged with cargo defining their specific functions remain elusive. : Here, we investigated the role of the chaperone Bcl2-associated anthogene 6 (BAG6, synonym Bat3) for the formation of pro- and anti-tumor EVs. EVs collected from wildtype cells and BAG6-deficient cells were characterized by mass spectrometry and RNAseq. Their tumorigenic potential was analyzed using the B-16V transplantation mouse melanoma model. : We demonstrate that EVs from B-16V cells inhibit lung metastasis associated with the mobilization of Ly6C patrolling monocytes. The formation of these anti-tumor-EVs was dependent on acetylation of p53 by the BAG6/CBP/p300-acetylase complex, followed by recruitment of components of the endosomal sorting complexes required for transport (ESCRT) via a P(S/T)AP double motif of BAG6. Genetic ablation of BAG6 and disruption of this pathway led to the release of a distinct EV subtype, which failed to suppress metastasis but recruited tumor-promoting neutrophils to the pre-metastatic niche. : We conclude that the BAG6/CBP/p300-p53 axis is a key pathway directing EV cargo loading and thus a potential novel microenvironmental therapeutic target.
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http://dx.doi.org/10.7150/thno.36378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735508PMC
September 2020

Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses.

EMBO J 2019 10 4;38(20):e102096. Epub 2019 Sep 4.

Institute of Molecular Oncology, Philipps-University, Marburg, Germany.

Engineered p53 mutant mice are valuable tools for delineating p53 functions in tumor suppression and cancer therapy. Here, we have introduced the R178E mutation into the Trp53 gene of mice to specifically ablate the cooperative nature of p53 DNA binding. Trp53 mice show no detectable target gene regulation and, at first sight, are largely indistinguishable from Trp53 mice. Surprisingly, stabilization of p53 in Mdm2 mice nevertheless triggers extensive apoptosis, indicative of residual wild-type activities. Although this apoptotic activity suffices to trigger lethality of Trp53 ;Mdm2 embryos, it proves insufficient for suppression of spontaneous and oncogene-driven tumorigenesis. Trp53 mice develop tumors indistinguishably from Trp53 mice and tumors retain and even stabilize the p53 protein, further attesting to the lack of significant tumor suppressor activity. However, Trp53 tumors exhibit remarkably better chemotherapy responses than Trp53 ones, resulting in enhanced eradication of p53-mutated tumor cells. Together, this provides genetic proof-of-principle evidence that a p53 mutant can be highly tumorigenic and yet retain apoptotic activity which provides a survival benefit in the context of cancer therapy.
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http://dx.doi.org/10.15252/embj.2019102096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792016PMC
October 2019

Comparative Transcriptomic Analysis of Temozolomide Resistant Primary GBM Stem-Like Cells and Recurrent GBM Identifies Up-Regulation of the Carbonic Anhydrase Gene as Resistance Factor.

Cancers (Basel) 2019 Jun 30;11(7). Epub 2019 Jun 30.

Department of Neurosurgery, UKGM, Philipps University Marburg, Baldingerstraße, 35033 Marburg, Germany.

About 95% of patients with Glioblastoma (GBM) show tumor relapse, leaving them with limited therapeutic options as recurrent tumors are most often resistant to the first line chemotherapy standard Temozolomide (TMZ). To identify molecular pathways involved in TMZ resistance, primary GBM Stem-like Cells (GSCs) were isolated, characterized, and selected for TMZ resistance in vitro. Subsequently, RNA sequencing analysis was performed and revealed a total of 49 differentially expressed genes (|log2-fold change| > 0.5 and adjusted -value < 0.1) in TMZ resistant stem-like cells compared to their matched DMSO control cells. Among up-regulated genes, we identified carbonic anhydrase 2 (CA2) as a candidate gene correlated with glioma malignancy and patient survival. Notably, we describe consistent up-regulation of CA2 not only in TMZ resistant GSCs on mRNA and protein level, but also in patient-matched clinical samples of first manifest and recurrent tumors. Co-treatment with the carbonic anhydrase inhibitor Acetazolamid (ACZ) sensitized cells to TMZ induced cell death. Cumulatively, our findings illustrate the potential of CA2 as a chemosensitizing target in recurrent GBM and provide a rationale for a therapy associated inhibition of CA2 to overcome TMZ induced chemoresistance.
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http://dx.doi.org/10.3390/cancers11070921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678269PMC
June 2019

Chemotherapeutic Drugs Inhibiting Topoisomerase 1 Activity Impede Cytokine-Induced and NF-κB p65-Regulated Gene Expression.

Cancers (Basel) 2019 Jun 25;11(6). Epub 2019 Jun 25.

Institute of Biochemistry, Justus Liebig University, D-35392 Giessen, Germany.

Inhibitors of DNA topoisomerase I (TOP1), an enzyme relieving torsional stress of DNA by generating transient single-strand breaks, are clinically used to treat ovarian, small cell lung and cervical cancer. As torsional stress is generated during transcription by progression of RNA polymerase II through the transcribed gene, we tested the effects of camptothecin and of the approved TOP1 inhibitors Topotecan and SN-38 on TNFα-induced gene expression. RNA-seq experiments showed that inhibition of TOP1 but not of TOP2 activity suppressed the vast majority of TNFα-triggered genes. The TOP1 effects were fully reversible and preferentially affected long genes. TNFα stimulation led to inducible recruitment of TOP1 to the gene body of , where its inhibition by camptothecin reduced transcription elongation and also led to altered histone H3 acetylation. Together, these data show that TOP1 inhibitors potently suppress expression of proinflammatory cytokines, a feature that may contribute to the increased infection risk occurring in tumor patients treated with these agents. On the other hand, TOP1 inhibitors could also be considered as a therapeutic option in order to interfere with exaggerated cytokine expression seen in several inflammatory diseases.
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http://dx.doi.org/10.3390/cancers11060883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627772PMC
June 2019

Specific allelic variants of SNPs in the and genes are associated with earlier tumor onset and progression in Caucasian breast cancer patients.

Oncotarget 2019 Mar 8;10(20):1975-1992. Epub 2019 Mar 8.

Department of Gynaecology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany.

Background: Genetic factors play a substantial role in breast cancer etiology. Genes encoding proteins that have key functions in the DNA damage response, such as p53 and its inhibitors MDM2 and MDMX, are most likely candidates to harbor allelic variants that influence breast cancer susceptibility. The aim of our study was to comprehensively analyze the impact of SNPs in the , , and genes in conjunction with mutational status regarding the onset and progression of breast cancer.

Methods: In specimen from 815 breast cancer patients, five SNPs within the selected genes were analyzed: - Arg72Pro (rs1042522), - SNP285 (rs2279744), SNP309 (rs117039649); - SNP31826 (rs1563828), and SNP34091 (rs4245739). Classification of the tumors was evaluated by histomorphology. Subtyping according hormone receptor status, HER2-status and proliferation rate enabled provision of the clinico-pathological surrogate of intrinsic subtypes.

Results: The homozygous C-allele of SNP285 was significantly associated with a younger age-at-diagnosis of 44.2 years, in contrast to G/G- and G/C-patients (62.4, 62.7 yrs., respectively; = 0.0007; log-Rank-test). In contrast, there was no difference regarding the age-at-diagnosis for patients with the respective genotypes of SNP309 ( = 0.799; log-Rank-test). In patients with estrogen receptor (ER)-positive and -mutated tumors, however, the T/T-genotype of the SNP309 was significantly associated with an earlier average age-at-diagnosis compared with T/G+G/G-patients (53.5 vs. 68.2 yrs; = 0.002; log-Rank-test). In the triple-negative subgroup, the G/G-patients had an average age-at-diagnosis of 51 years compared with 63 years for SNP309T carriers ( = 0.004; log-Rank-test) indicating a susceptibility of the G/G genotype for the development of triple negative breast cancer. Patients with the A/A-genotype of SNP31826 with ER-negative tumors were diagnosed 11 years earlier compared with patients and ER-positive tumors (53.2 vs. 64.4 yrs; = 0.025, log-Rank-test). Furthermore, in luminal B-like patients (HER2-independent) the C/C-genotype of SNP34091 was significantly correlated with a decreased event-free survival compared with the A/A-genotype ( < 0.001; log-Rank-test).

Conclusions: We showed that SNPs in the and genes affect at least in part the onset and progression of breast cancer dependent on the ER-status. Our findings provide further evidence for the distinct etiological pathways in ER-negative and ER-positive breast cancers.
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http://dx.doi.org/10.18632/oncotarget.26768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443004PMC
March 2019

SMARCAD1 ATPase activity is required to silence endogenous retroviruses in embryonic stem cells.

Nat Commun 2019 03 22;10(1):1335. Epub 2019 Mar 22.

Institute of Molecular Biology and Tumour Research, Philipps University Marburg, Marburg, 35043, Germany.

Endogenous retroviruses (ERVs) can confer benefits to their host but present a threat to genome integrity if not regulated correctly. Here we identify the SWI/SNF-like remodeler SMARCAD1 as a key factor in the control of ERVs in embryonic stem cells. SMARCAD1 is enriched at ERV subfamilies class I and II, particularly at active intracisternal A-type particles (IAPs), where it preserves repressive histone methylation marks. Depletion of SMARCAD1 results in de-repression of IAPs and adjacent genes. Recruitment of SMARCAD1 to ERVs is dependent on KAP1, a central component of the silencing machinery. SMARCAD1 and KAP1 occupancy at ERVs is co-dependent and requires the ATPase function of SMARCAD1. Our findings uncover a role for the enzymatic activity of SMARCAD1 in cooperating with KAP1 to silence ERVs. This reveals ATP-dependent chromatin remodeling as an integral step in retrotransposon regulation in stem cells and advances our understanding of the mechanisms driving heterochromatin establishment.
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http://dx.doi.org/10.1038/s41467-019-09078-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430823PMC
March 2019