Publications by authors named "Sebastian Oeck"

22 Publications

  • Page 1 of 1

Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis.

Cell Rep 2021 Nov;37(8):110056

Department of Medical Oncology, West German Cancer Center, University Hospital Essen at the University Duisburg-Essen, Duisburg, Germany; German Cancer Consortium (DKTK) partner site Essen, Essen, Germany. Electronic address:

Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.
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http://dx.doi.org/10.1016/j.celrep.2021.110056DOI Listing
November 2021

NTRK1/TrkA Signaling in Neuroblastoma Cells Induces Nuclear Reorganization and Intra-Nuclear Aggregation of Lamin A/C.

Cancers (Basel) 2021 Oct 21;13(21). Epub 2021 Oct 21.

West German Cancer Center, Department of Medical Oncology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany.

(1) Background: Neuroblastomas (NBs) are the most common extracranial solid tumors of children. The amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB aggressiveness, while high expression of the neurotrophin receptor NTRK1/TrkA is associated with mild disease courses. The molecular effects of NTRK1 signaling in MYCN-amplified NB, however, are still poorly understood and require elucidation. (2) Methods: Inducible NTRK1 expression was realized in four NB cell lines with (IMR5, NGP) or without MYCN amplification (SKNAS, SH-SY5Y). Proteome and phosphoproteome dynamics upon NTRK1 activation by its ligand, NGF, were analyzed in a time-dependent manner in IMR5 cells. Target validation by immunofluorescence staining and automated image processing was performed using the three other NB cell lines. (3) Results: In total, 230 proteins and 134 single phosphorylated class I phosphosites were found to be significantly regulated upon NTRK1 activation. Among known NTRK1 targets, Stathmin and the neurosecretory protein VGF were recovered. Additionally, we observed the upregulation and phosphorylation of Lamin A/C (LMNA) that accumulated inside nuclear foci. (4) Conclusions: We provide a comprehensive picture of NTRK1-induced proteome and phosphoproteome dynamics. The phosphorylation of LMNA within nucleic aggregates was identified as a prominent feature of NTRK1 signaling independent of the MYCN status of NB cells.
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http://dx.doi.org/10.3390/cancers13215293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8582546PMC
October 2021

HER2 mediates clinical resistance to the KRAS inhibitor sotorasib, which is overcome by co-targeting SHP2.

Eur J Cancer 2021 Oct 26;159:16-23. Epub 2021 Oct 26.

Laboratory of Molecular Oncology, Department of Medical Oncology, West German Cancer Center, University Hospital Essen, Essen, Germany.

Introduction: Mutant RAS guanosine triphosphate hydrolases (GTPases) are key oncogenic drivers in many cancers. The KRAS variant has recently become targetable by a new drug class specifically locking KRAS in its inactive guanosine diphosphate (GDP)-bound state. Clinical activity was demonstrated in patients with advanced lung cancers harbouring KRAS mutations but was limited by the development of resistance.

Methods: A biopsy from progressing lung cancer of a patient treated with the KRAS inhibitor sotorasib was obtained, and the underlying resistance factors were analysed. Mechanistic studies were performed in vitro and in vivo to uncover strategies to overcome resistance to KRAS inhibition.

Results: We demonstrated acquisition of HER2 copy number gain and KRAS mutation retention in the post-progression biopsy. To explore HER2 gain as the relevant resistance mechanism, we generated KRAS lung cancer models overexpressing HER2. MAPK pathway signalling remained active despite KRAS inhibitor treatment. Combined pharmacological inhibition of KRAS and SHP2 synergistically overcame HER2-mediated resistance in vitro and in vivo.

Conclusions: These findings establish HER2 copy number gain as a clinically relevant mechanism of resistance to pharmacological KRAS inhibition that can be overcome by co-targeting SHP2.
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http://dx.doi.org/10.1016/j.ejca.2021.10.003DOI Listing
October 2021

Differential immunomodulatory effect of PARP inhibition in BRCA1 deficient and competent tumor cells.

Biochem Pharmacol 2021 02 4;184:114359. Epub 2020 Dec 4.

Department of Cellular and Molecular Medicine, University of Arizona Cancer Center College, Tucson, AZ, USA. Electronic address:

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors (PARPi) have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARPi induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin-bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms.
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http://dx.doi.org/10.1016/j.bcp.2020.114359DOI Listing
February 2021

Streamlining Quantitative Analysis of Long RNA Sequencing Reads.

Int J Mol Sci 2020 Oct 1;21(19). Epub 2020 Oct 1.

Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany.

Transcriptome analyses allow for linking RNA expression profiles to cellular pathways and phenotypes. Despite improvements in sequencing methodology, whole transcriptome analyses are still tedious, especially for methodologies producing long reads. Currently, available data analysis software often lacks cost- and time-efficient workflows. Although kit-based workflows and benchtop platforms for RNA sequencing provide software options, e.g., cloud-based tools to analyze basecalled reads, quantitative, and easy-to-use solutions for transcriptome analysis, especially for non-human data, are missing. We therefore developed a user-friendly tool, termed Alignator, for rapid analysis of long RNA reads requiring only FASTQ files and an Ensembl cDNA database reference. After successful mapping, Alignator generates quantitative information for each transcript and provides a table in which sequenced and aligned RNA are stored for further comparative analyses.
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http://dx.doi.org/10.3390/ijms21197259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584020PMC
October 2020

Hypoxia Induces Resistance to EGFR Inhibitors in Lung Cancer Cells via Upregulation of FGFR1 and the MAPK Pathway.

Cancer Res 2020 11 1;80(21):4655-4667. Epub 2020 Sep 1.

Department of Therapeutic Radiology, Yale University School of Medicine. New Haven, Connecticut.

Development of resistance remains the key obstacle to the clinical efficacy of EGFR tyrosine kinase inhibitors (TKI). Hypoxia is a key microenvironmental stress in solid tumors associated with acquired resistance to conventional therapy. Consistent with our previous studies, we show here that long-term, moderate hypoxia promotes resistance to the EGFR TKI osimertinib (AZD9291) in the non-small cell lung cancer (NSCLC) cell line H1975, which harbors two EGFR mutations including T790M. Hypoxia-induced resistance was associated with development of epithelial-mesenchymal transition (EMT) coordinated by increased expression of ZEB-1, an EMT activator. Hypoxia induced increased fibroblast growth factor receptor 1 (FGFR1) expression in NSCLC cell lines H1975, HCC827, and YLR086, and knockdown of FGFR1 attenuated hypoxia-induced EGFR TKI resistance in each line. Upregulated expression of FGFR1 by hypoxia was mediated through the MAPK pathway and attenuated induction of the proapoptotic factor BIM. Consistent with this, inhibition of FGFR1 function by the selective small-molecule inhibitor BGJ398 enhanced EGFR TKI sensitivity and promoted upregulation of BIM levels. Furthermore, inhibition of MEK activity by trametinib showed similar effects. In tumor xenografts in mice, treatment with either BGJ398 or trametinib enhanced response to AZD9291 and improved survival. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased expression of FGFR1. The combination of EGFR TKI and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLC. SIGNIFICANCE: Hypoxia-induced resistance to EGFR TKI is driven by overexpression of FGFR1 to sustain ERK signaling, where a subsequent combination of EGFR TKI with FGFR1 inhibitors or MEK inhibitors reverses this resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4655/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642024PMC
November 2020

Oncometabolites suppress DNA repair by disrupting local chromatin signalling.

Nature 2020 06 3;582(7813):586-591. Epub 2020 Jun 3.

Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR) and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.
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http://dx.doi.org/10.1038/s41586-020-2363-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319896PMC
June 2020

Mitochondrial DNA Stress Signalling Protects the Nuclear Genome.

Nat Metab 2019 12 9;1(12):1209-1218. Epub 2019 Dec 9.

Salk Institute for Biological Studies, La Jolla, CA, USA.

The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology. We showed previously that instability and cytoplasmic release of mtDNA activates the cGAS-STING-TBK1 pathway resulting in interferon stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs, that includes , remains activated by the unphosphorylated form of ISGF3 (U-ISGF3) that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS-STING-dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin . Finally, mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting mtDNA is a genotoxic stress sentinel.
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http://dx.doi.org/10.1038/s42255-019-0150-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213273PMC
December 2019

Proton Irradiation Increases the Necessity for Homologous Recombination Repair Along with the Indispensability of Non-Homologous End Joining.

Cells 2020 04 5;9(4). Epub 2020 Apr 5.

Institute of Cell Biology (Cancer Research), University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany.

Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.
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http://dx.doi.org/10.3390/cells9040889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226794PMC
April 2020

Optimizing biodegradable nanoparticle size for tissue-specific delivery.

J Control Release 2019 11 22;314:92-101. Epub 2019 Oct 22.

Department of Biomedical Engineering, Yale University, New Haven, CT, 06511, USA; Department of Chemical & Environmental Engineering, Yale University, New Haven, CT, 06511, USA; Department of Physiology, Yale University, New Haven, CT, 06511, USA. Electronic address:

Nanoparticles (NPs) are promising vehicles for drug delivery because of their potential to target specific tissues [1]. Although it is known that NP size plays a critical role in determining their biological activity, there are few quantitative studies of the role of NP size in determining biodistribution after systemic administration. Here, we engineered fluorescent, biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs in a range of sizes (120-440nm) utilizing a microfluidic platform and used these NPs to determine the effect of diameter on bulk tissue and cellular distribution after systemic administration. We demonstrate that small NPs (∼120nm) exhibit enhanced uptake in bulk lung and bone marrow, while larger NPs are sequestered in the liver and spleen. We also show that small NPs (∼120nm) access specific alveolar cell populations and hematopoietic stem and progenitor cells more readily than larger NPs. Our results suggest that size of PLGA NPs can be used to tune delivery to certain tissues and cell populations in vivo.
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http://dx.doi.org/10.1016/j.jconrel.2019.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909251PMC
November 2019

Cediranib suppresses homology-directed DNA repair through down-regulation of BRCA1/2 and RAD51.

Sci Transl Med 2019 05;11(492)

Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA.

Combining the anti-angiogenic agent cediranib with the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib improves progression-free survival compared to olaparib alone in ovarian cancer patients through an unknown mechanism. PARP inhibitors are used primarily in the treatment of patients with DNA repair-associated (BRCA1/2) mutated cancers because these mutations cause a deficit in homology-directed DNA repair (HDR) that confers sensitivity to these agents. However, the combination of cediranib and olaparib was effective in patients without BRCA1/2 mutations. We report here that cediranib confers sensitivity to olaparib by down-regulating HDR in tumor cells. This occurs partially as a result of cediranib inducing hypoxia, which suppresses expression of the HDR factors BRCA1/2 and RAD51 recombinase (RAD51). However, we also observed that cediranib has a direct effect on HDR independent of its ability to induce tumor hypoxia. This direct effect occurs through platelet-derived growth factor receptor (PDGFR) inhibition, activation of protein phosphatase 2A (PP2A), and E2F transcription factor 4 (E2F4)/RB transcriptional corepressor like 2 (RB2/p130)-mediated repression of and gene expression. This down-regulation was seen in mouse tumor xenografts but not in mouse bone marrow, providing a therapeutic window for combining cediranib and olaparib in cancer therapy. Our work reveals a treatment strategy by which DNA repair can be manipulated in human tumors to induce synthetic lethality, broadening the potential therapeutic scope of cediranib based on its activity as a DNA repair inhibitor.
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http://dx.doi.org/10.1126/scitranslmed.aav4508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626544PMC
May 2019

High-throughput Evaluation of Protein Migration and Localization after Laser Micro-Irradiation.

Sci Rep 2019 02 28;9(1):3148. Epub 2019 Feb 28.

Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, 06520, USA.

DNA- and histone-related research frequently comprises the quantitative analysis of protein modifications, such as histone phosphorylation. Analysis of accumulation and disappearance of protein foci are used to monitor DNA damage and repair kinetics. If the protein of interest doesn't accumulate in foci, laser micro-irradiation of single nuclei provides an alternative method to monitor DNA repair proteins and histone dynamics at the DNA damage site. We have developed an automated evaluation tool for standardized, high-throughput analysis of micro-irradiated cells featuring single cell background subtraction and detection across multiple fluorescence channels, allowing for robust statistics.
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http://dx.doi.org/10.1038/s41598-019-39760-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395640PMC
February 2019

Relating Linear Energy Transfer to the Formation and Resolution of DNA Repair Foci After Irradiation with Equal Doses of X-ray Photons, Plateau, or Bragg-Peak Protons.

Int J Mol Sci 2018 Nov 28;19(12). Epub 2018 Nov 28.

Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Virchowstrasse 173, 45122 Essen, Germany.

Proton beam therapy is increasingly applied for the treatment of human cancer, as it promises to reduce normal tissue damage. However, little is known about the relationship between linear energy transfer (LET), the type of DNA damage, and cellular repair mechanisms, particularly for cells irradiated with protons. We irradiated cultured cells delivering equal doses of X-ray photons, Bragg-peak protons, or plateau protons and used this set-up to quantitate initial DNA damage (mainly DNA double strand breaks (DSBs)), and to analyze kinetics of repair by detecting γH2A.X or 53BP1 using immunofluorescence. The results obtained validate the reliability of our set-up in delivering equal radiation doses under all conditions employed. Although the initial numbers of γH2A.X and 53BP1 foci scored were similar under the different irradiation conditions, it was notable that the maximum foci level was reached at 60 min after irradiation with Bragg-peak protons, as compared to 30 min for plateau protons and photons. Interestingly, Bragg-peak protons induced larger and irregularly shaped γH2A.X and 53BP1 foci. Additionally, the resolution of these foci was delayed. These results suggest that Bragg-peak protons induce DNA damage of increased complexity which is difficult to process by the cellular repair apparatus.
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http://dx.doi.org/10.3390/ijms19123779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320817PMC
November 2018

Restraining Akt1 Phosphorylation Attenuates the Repair of Radiation-Induced DNA Double-Strand Breaks and Reduces the Survival of Irradiated Cancer Cells.

Int J Mol Sci 2018 Jul 31;19(8). Epub 2018 Jul 31.

Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, University Hospital Essen Virchowstrasse 173, 45147 Essen, Germany.

The survival kinase protein kinase B (Akt) participates in the regulation of essential subcellular processes, e.g., proliferation, growth, survival, and apoptosis, and has a documented role in promoting resistance against genotoxic stress including radiotherapy, presumably by influencing the DNA damage response and DNA double-strand break (DSB) repair. However, its exact role in DSB repair requires further elucidation. We used a genetic approach to explore the consequences of impaired phosphorylation of Akt1 at one or both of its key phosphorylation sites, Threonine 308 (T308) or Serine 473 (S473), on DSB repair and radiosensitivity to killing. Therefore, we overexpressed either the respective single or the double phosphorylation-deficient mutants (Akt1-T308A, Akt1-S473A, or Akt1-T308A/S473A) in TRAMPC1 murine prostate cancer cells (TrC1) and measured the DSB repair kinetics and clonogenic cell survival upon irradiation. Only the expression of the Akt1-T308A/S473A induced a significant delay in the kinetics of DSB repair in irradiated TrC1 as determined by the γH2A.X (H2A histone family, member X) assay and the neutral comet assay, respectively. Moreover, Akt1-T308A/S473A-expressing cells were characterized by increased radiosensitivity compared to Akt1-WT (wild type)-expressing cells in long-term colony formation assays. Our data reveal that Akt1's activation state is important for the cellular radiation response, presumably by modulating the phosphorylation of effector proteins involved in the regulation of DSB repair.
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http://dx.doi.org/10.3390/ijms19082233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121313PMC
July 2018

Krebs-cycle-deficient hereditary cancer syndromes are defined by defects in homologous-recombination DNA repair.

Nat Genet 2018 08 16;50(8):1086-1092. Epub 2018 Jul 16.

Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA.

The hereditary cancer syndromes hereditary leiomyomatosis and renal cell cancer (HLRCC) and succinate dehydrogenase-related hereditary paraganglioma and pheochromocytoma (SDH PGL/PCC) are linked to germline loss-of-function mutations in genes encoding the Krebs cycle enzymes fumarate hydratase and succinate dehydrogenase, thus leading to elevated levels of fumarate and succinate, respectively. Here, we report that fumarate and succinate both suppress the homologous recombination (HR) DNA-repair pathway required for the resolution of DNA double-strand breaks (DSBs) and for the maintenance of genomic integrity, thus rendering tumor cells vulnerable to synthetic-lethal targeting with poly(ADP)-ribose polymerase (PARP) inhibitors. These results identify HLRCC and SDH PGL/PCC as familial DNA-repair deficiency syndromes, providing a mechanistic basis to explain their cancer predisposition and suggesting a potentially therapeutic approach for advanced HLRCC and SDH PGL/PCC, both of which are incurable when metastatic.
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http://dx.doi.org/10.1038/s41588-018-0170-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072579PMC
August 2018

Hypoxia Promotes Resistance to EGFR Inhibition in NSCLC Cells via the Histone Demethylases, LSD1 and PLU-1.

Mol Cancer Res 2018 10 22;16(10):1458-1469. Epub 2018 Jun 22.

Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut.

The development of small-molecule tyrosine kinase inhibitors (TKI) specific for epidermal growth factor receptors (EGFR) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key microenvironmental stress in solid tumors that is associated with poor prognosis due, in part, to acquired resistance to conventional therapy. This study documents that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line HCC827, which harbors an activating EGFR mutation. Following hypoxic growth conditions, HCC827 cells treated with gefitinib upregulated N-cadherin, fibronectin, and vimentin expression and downregulated E-cadherin, characteristic of an epithelial-mesenchymal transition (EMT), which prior studies have linked to EGFR TKI resistance. Mechanistically, knockdown of the histone demethylases, LSD1 and PLU-1, prevented and reversed hypoxia-induced gefitinib resistance, with inhibition of the associated EMT, suggesting that LSD1 and PLU-1 play key roles in hypoxia-induced gefitinib resistance and EMT. Moreover, hypoxia-treated HCC827 cells demonstrated more aggressive tumor growth compared with cells grown in normoxia, but inhibition of LSD1 function by shRNA-mediated knockdown or by the small-molecular inhibitor SP2509 suppressed tumor growth and enhanced gefitinib response These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through epigenetic change and coordination of EMT in NSCLC. This study suggests that combination of therapy with EGFR TKIs and LSD1 inhibitors may offer an attractive therapeutic strategy for NSCLCs. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170700PMC
October 2018

PTEN Regulates Nonhomologous End Joining By Epigenetic Induction of NHEJ1/XLF.

Mol Cancer Res 2018 08 8;16(8):1241-1254. Epub 2018 May 8.

Department of Genetics, Yale University, New Haven, Connecticut.

DNA double-strand breaks (DSB) are the most cytotoxic DNA lesions, and up to 90% of DSBs require repair by nonhomologous end joining (NHEJ). Functional and genomic analyses of patient-derived melanomas revealed that PTEN loss is associated with NHEJ deficiency. In PTEN-null melanomas, PTEN complementation rescued the NHEJ defect; conversely, suppression of PTEN compromised NHEJ. Mechanistic studies revealed that PTEN promotes NHEJ through direct induction of expression of XRCC4-like factor (/XLF), which functions in DNA end bridging and ligation. PTEN was found to occupy the gene promoter and to recruit the histone acetyltransferases, PCAF and CBP, inducing XLF expression. This recruitment activity was found to be independent of its phosphatase activity, but dependent on K128, a site of regulatory acetylation on PTEN. These findings define a novel function for PTEN in regulating NHEJ DSB repair, and therefore may assist in the design of individualized strategies for cancer therapy. PTEN is the second most frequently lost tumor suppressor gene. Here it is demonstrated that PTEN has a direct and novel regulatory role in NHEJ, a key DNA repair pathway in response to radiation and chemotherapy. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072556PMC
August 2018

New Insights into Protein Kinase B/Akt Signaling: Role of Localized Akt Activation and Compartment-Specific Target Proteins for the Cellular Radiation Response.

Cancers (Basel) 2018 Mar 18;10(3). Epub 2018 Mar 18.

Institute of Cell Biology (Cancer Research), University of Duisburg-Essen Medical School, 45122 Essen, Germany.

Genetic alterations driving aberrant activation of the survival kinase Protein Kinase B (Akt) are observed with high frequency during malignant transformation and cancer progression. Oncogenic gene mutations coding for the upstream regulators or Akt, e.g., growth factor receptors, RAS and phosphatidylinositol-3-kinase (PI3K), or for one of the three Akt isoforms as well as loss of the tumor suppressor Phosphatase and Tensin Homolog on Chromosome Ten (PTEN) lead to constitutive activation of Akt. By activating Akt, these genetic alterations not only promote growth, proliferation and malignant behavior of cancer cells by phosphorylation of various downstream signaling molecules and signaling nodes but can also contribute to chemo- and radioresistance in many types of tumors. Here we review current knowledge on the mechanisms dictating Akt's activation and target selection including the involvement of miRNAs and with focus on compartmentalization of the signaling network. Moreover, we discuss recent advances in the cross-talk with DNA damage response highlighting nuclear Akt target proteins with potential involvement in the regulation of DNA double strand break repair.
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http://dx.doi.org/10.3390/cancers10030078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876653PMC
March 2018

Epigenetic Mechanisms of ATM Activation after Helicobacter pylori Infection.

Am J Pathol 2018 02 9;188(2):329-335. Epub 2017 Nov 9.

Clinical Pharmacology and Gastroenterology Unit, Sao Francisco University Medical School, Bragança Paulista, São Paulo, Brazil. Electronic address:

Gastric cancer (GC) is the second leading cause of cancer-related mortality worldwide. The disease develops from the accumulation of several genetic and epigenetic changes. Among other risk factors, Helicobacter pylori infection is considered the main driving factor of GC development. H. pylori infection increases DNA damage levels and leads to epigenetic dysregulation, which may favor gastric carcinogenesis. An early step in double-strand break repair is the recruitment of ataxia-telangiectasia mutated serine/threonine kinase (ATM) to the damaged site, where it plays a key role in advancing the DNA damage checkpoint process. H. pylori infection has been associated with the introduction of double-strand breaks in epithelial cells, triggering damage signaling and repair response involving ATM. Thus, the current study analyzed the effect of H. pylori infection on the DNA damage response sensor, ATM, in gastric epithelial cells and in biopsy specimens from patients with GC. In this study, we identified that H. pylori infection stimulated DNA damage, and therefore induced ATM in a virulence factor-dependent manner. In addition, we found that H. pylori might activate ATM through histone H3 and H4 hyperacetylation and DNA promoter hypomethylation. Our findings show a mechanism associating ATM signaling induction with H. pylori infection.
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http://dx.doi.org/10.1016/j.ajpath.2017.10.005DOI Listing
February 2018

The Focinator v2-0 - Graphical Interface, Four Channels, Colocalization Analysis and Cell Phase Identification.

Radiat Res 2017 07 11;188(1):114-120. Epub 2017 May 11.

Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Virchowstrasse 173, 45122 Essen, Germany.

The quantitative analysis of foci plays an important role in various cell biological methods. In the fields of radiation biology and experimental oncology, the effect of ionizing radiation, chemotherapy or molecularly targeted drugs on DNA damage induction and repair is frequently performed by the analysis of protein clusters or phosphorylated proteins recruited to so called repair foci at DNA damage sites, involving for example γ-H2A.X, 53BP1 or RAD51. We recently developed "The Focinator" as a reliable and fast tool for automated quantitative and qualitative analysis of nuclei and DNA damage foci. The refined software is now even more user-friendly due to a graphical interface and further features. Thus, we included an R-script-based mode for automated image opening, file naming, progress monitoring and an error report. Consequently, the evaluation no longer required the attendance of the operator after initial parameter definition. Moreover, the Focinator v2-0 is now able to perform multi-channel analysis of four channels and evaluation of protein-protein colocalization by comparison of up to three foci channels. This enables for example the quantification of foci in cells of a specific cell cycle phase.
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http://dx.doi.org/10.1667/RR14746.1DOI Listing
July 2017

The Focinator - a new open-source tool for high-throughput foci evaluation of DNA damage.

Radiat Oncol 2015 Aug 4;10:163. Epub 2015 Aug 4.

Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Virchowstrasse 173, 45122, Essen, Germany.

Background: The quantitative analysis of foci plays an important role in many cell biological methods such as counting of colonies or cells, organelles or vesicles, or the number of protein complexes. In radiation biology and molecular radiation oncology, DNA damage and DNA repair kinetics upon ionizing radiation (IR) are evaluated by counting protein clusters or accumulations of phosphorylated proteins recruited to DNA damage sites. Consistency in counting and interpretation of foci remains challenging. Many current software solutions describe instructions for time-consuming and error-prone manual analysis, provide incomplete algorithms for analysis or are expensive. Therefore, we aimed to develop a tool for costless, automated, quantitative and qualitative analysis of foci.

Methods: For this purpose we integrated a user-friendly interface into ImageJ and selected parameters to allow automated selection of regions of interest (ROIs) depending on their size and circularity. We added different export options and a batch analysis. The use of the Focinator was tested by analyzing γ-H2.AX foci in murine prostate adenocarcinoma cells (TRAMP-C1) at different time points after IR with 0.5 to 3 Gray (Gy). Additionally, measurements were performed by users with different backgrounds and experience.

Results: The Focinator turned out to be an easily adjustable tool for automation of foci counting. It significantly reduced the analysis time of radiation-induced DNA-damage foci. Furthermore, different user groups were able to achieve a similar counting velocity. Importantly, there was no difference in nuclei detection between the Focinator and ImageJ alone.

Conclusions: The Focinator is a costless, user-friendly tool for fast high-throughput evaluation of DNA repair foci. The macro allows improved foci evaluation regarding accuracy, reproducibility and analysis speed compared to manual analysis. As innovative option, the macro offers a combination of multichannel evaluation including colocalization analysis and the possibility to run all analyses in a batch mode.
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http://dx.doi.org/10.1186/s13014-015-0453-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4554354PMC
August 2015

Covalent-Allosteric Kinase Inhibitors.

Angew Chem Int Ed Engl 2015 Aug 25;54(35):10313-6. Epub 2015 Jun 25.

Technische Universität Dortmund, Fakultät für Chemie und Chemische Biologie, Otto-Hahn-Strasse 6, 44227 Dortmund (Germany).

Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-dependent covalent-allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research.
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http://dx.doi.org/10.1002/anie.201502142DOI Listing
August 2015
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