Publications by authors named "Daniel Nettersheim"

43 Publications

The component of the mA writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors.

J Exp Clin Cancer Res 2021 Aug 25;40(1):268. Epub 2021 Aug 25.

Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / [email protected] (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal.

Background: Germ cell tumors (GCTs) are developmental cancers, tightly linked to embryogenesis and germ cell development. The recent and expanding field of RNA modifications is being increasingly implicated in such molecular events, as well as in tumor progression and resistance to therapy, but still rarely explored in GCTs. In this work, and as a follow-up of our recent study on this topic in TGCT tissue samples, we aim to investigate the role of N6-methyladenosine (mA), the most abundant of such modifications in mRNA, in in vitro and in vivo models representative of such tumors.

Methods: Four cell lines representative of GCTs (three testicular and one mediastinal), including an isogenic cisplatin resistant subline, were used. CRISPR/Cas9-mediated knockdown of VIRMA was established and the chorioallantoic membrane assay was used to study its phenotypic effect in vivo.

Results: We demonstrated the differential expression of the various mA writers, readers and erasers in GCT cell lines representative of the major classes of these tumors, seminomas and non-seminomas, and we evidenced changes occurring upon differentiation with all-trans retinoic acid treatment. We showed differential expression also among cells sensitive and resistant to cisplatin treatment, implicating these players in acquisition of cisplatin resistant phenotype. Knockdown of VIRMA led to disruption of the remaining methyltransferase complex and decrease in mA abundance, as well as overall reduced tumor aggressiveness (with decreased cell viability, tumor cell proliferation, migration, and invasion) and increased sensitivity to cisplatin treatment, both in vitro and confirmed in vivo. Enhanced response to cisplatin after VIRMA knockdown was related to significant increase in DNA damage (with higher γH2AX and GADD45B levels) and downregulation of XLF and MRE11.

Conclusions: VIRMA has an oncogenic role in GCTs confirming our previous tissue-based study and is further involved in response to cisplatin by interfering with DNA repair. These data contribute to our better understanding of the emergence of cisplatin resistance in GCTs and support recent attempts to therapeutically target elements of the mA writer complex.
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http://dx.doi.org/10.1186/s13046-021-02072-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390281PMC
August 2021

The developmental origin of cancers defines basic principles of cisplatin resistance.

Cancer Lett 2021 Oct 25;519:199-210. Epub 2021 Jul 25.

Department of Urology, Urological Research Laboratory, Translational UroOncology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University, Universitätsstraße 1, 40225 Düsseldorf, Germany. Electronic address:

Cisplatin-based chemotherapy has been used for more than four decades as a standard therapeutic option in several tumor entities. However, being a multifaceted and heterogeneous phenomenon, inherent or acquired resistance to cisplatin remains a major obstacle during the treatment of several solid malignancies and inevitably results in disease progression. Hence, we felt there was an urgent need to evaluate common mechanisms between multifarious cancer entities to identify patient-specific therapeutic strategies. We found joint molecular and (epi)genetic resistance mechanisms and specific cisplatin-induced mutational signatures that depended on the developmental origin (endo-, meso-, ectoderm) of the tumor tissue. Based on the findings of thirteen tumor entities, we identified three resistance groups, where Group 1 (endodermal origin) prominently indicates NRF2-pathway activation, Group 2 (mesodermal origin, primordial germ cells) shares elevated DNA repair mechanisms and decreased apoptosis induction, and Group 3 (ectodermal and paraxial mesodermal origin) commonly presents deregulated apoptosis induction and alternating pathways as the main cisplatin-induced resistance mechanisms. This review further proposes potential and novel therapeutic strategies to improve the outcome of cisplatin-based chemotherapy.
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http://dx.doi.org/10.1016/j.canlet.2021.07.037DOI Listing
October 2021

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas.

Mol Oncol 2021 Jul 22. Epub 2021 Jul 22.

Department of Urology, Urological Research Laboratory, Translational UroOncology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Germany.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.
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http://dx.doi.org/10.1002/1878-0261.13066DOI Listing
July 2021

On the origin of germ cell neoplasia in situ: Dedifferentiation of human adult Sertoli cells in cross talk with seminoma cells in vitro.

Neoplasia 2021 07 18;23(7):731-742. Epub 2021 Jun 18.

Department of Internal Medicine, Member of the German Center for Lung Research (DZL), Germany; Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Member of the DZL, Member of CPI, Bad Nauheim, Germany; Institute for Lung Health (ILH), Justus-Liebig-University, Giessen, Germany. Electronic address:

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.
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http://dx.doi.org/10.1016/j.neo.2021.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8233172PMC
July 2021

Molecular and epigenetic pathogenesis of germ cell tumors.

Asian J Urol 2021 Apr 30;8(2):144-154. Epub 2020 May 30.

Department of Urology, Urological Research Lab, Translational UroOncology, University Hospital Düsseldorf, Düsseldorf, Germany.

The development of germ cell tumors (GCTs) is a unique pathogenesis occurring at an early developmental stage during specification, migration or colonization of primordial germ cells (PGCs) in the genital ridge. Since driver mutations could not be identified so far, the involvement of the epigenetic machinery during the pathogenesis seems to play a crucial role. Currently, it is investigated whether epigenetic modifications occurring between the omnipotent two-cell stage and the pluripotent implanting PGCs might result in disturbances eventually leading to GCTs. Although progress in understanding epigenetic mechanisms during PGC development is ongoing, little is known about the complete picture of its involvement during GCT development and eventual classification into clinical subtypes. This review will shed light into the current knowledge of the complex epigenetic and molecular contribution during pathogenesis of GCTs by emphasizing on early developmental stages until arrival of late PGCs in the gonads. We questioned how misguided migrating and/or colonizing PGCs develop to either type I or type II GCTs. Additionally, we asked how pluripotency can be regulated during PGC development and which epigenetic changes contribute to GCT pathogenesis. We propose that SOX2 and SOX17 determine either embryonic stem cell-like (embryonal carcinoma) or PGC-like cell fate (seminoma). Finally, we suggest that factors secreted by the microenvironment, BMPs and BMP inhibiting molecules, dictate the fate decision of germ cell neoplasia (into seminoma and embryonal carcinoma) and seminomas (into embryonal carcinoma or extraembryonic lineage), indicating an important role of the microenvironment on GCT plasticity.
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http://dx.doi.org/10.1016/j.ajur.2020.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099689PMC
April 2021

The pioneer and differentiation factor FOXA2 is a key driver of yolk-sac tumour formation and a new biomarker for paediatric and adult yolk-sac tumours.

J Cell Mol Med 2021 02 14;25(3):1394-1405. Epub 2021 Jan 14.

Department of Urology, Urological Research Lab, Translational UroOncology, University Hospital Düsseldorf, Düsseldorf, Germany.

Yolk-sac tumours (YSTs), a germ cell tumour subtype, occur in newborns and infants as well as in young adults of age 14-44 years. In clinics, adult patients with YSTs face a poor prognosis, as these tumours are often therapy-resistant and count for many germ cell tumour related deaths. So far, the molecular and (epi)genetic mechanisms that control development of YST are far from being understood. We deciphered the molecular and (epi)genetic mechanisms regulating YST formation by meta-analysing high-throughput data of gene and microRNA expression, DNA methylation and mutational burden. We validated our findings by qRT-PCR and immunohistochemical analyses of paediatric and adult YSTs. On a molecular level, paediatric and adult YSTs were nearly indistinguishable, but were considerably different from embryonal carcinomas, the stem cell precursor of YSTs. We identified FOXA2 as a putative key driver of YST formation, subsequently inducing AFP, GPC3, APOA1/APOB, ALB and GATA3/4/6 expression. In YSTs, WNT-, BMP- and MAPK signalling-related genes were up-regulated, while pluripotency- and (primordial) germ cell-associated genes were down-regulated. Expression of FOXA2 and related key factors seems to be regulated by DNA methylation, histone methylation / acetylation and microRNAs. Additionally, our results highlight FOXA2 as a promising new biomarker for paediatric and adult YSTs.
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http://dx.doi.org/10.1111/jcmm.16222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875904PMC
February 2021

Filamin A Orchestrates Cytoskeletal Structure, Cell Migration and Stem Cell Characteristics in Human Seminoma TCam-2 Cells.

Cells 2020 11 30;9(12). Epub 2020 Nov 30.

Anatomy III, Cell Biology, Biomedical Center, Ludwig Maximillian University of Munich, 82152 Planegg-Martinsried, Germany.

Filamins are large dimeric F-actin cross-linking proteins, crucial for the mechanosensitive properties of a number of cell types. Due to their interaction with a variety of different proteins, they exert important regulatory functions. However, in the human testis the role of filamins has been insufficiently explored. Immunohistochemical staining of human testis samples identified filamin A (FLNA) in spermatogonia and peritubular myoid cells. Investigation of different testicular tumor samples indicated that seminoma also express FLNA. Moreover, mass spectrometric analyses identified FLNA as one of the most abundant proteins in human seminoma TCam-2 cells. We therefore focused on FLNA in TCam-2 cells, and identified by co-immunoprecipitation LAD1, RUVBL1 and DAZAP1, in addition to several cytoskeletal proteins, as interactors of FLNA. To study the role of FLNA in TCam-2 cells, we generated FLNA-deficient cells using the CRISPR/Cas9 system. Loss of FLNA causes an irregular arrangement of the actin cytoskeleton and mechanical instability, impaired adhesive properties and disturbed migratory behavior. Furthermore, transcriptional activity of typical stem cell factors is increased in the absence of FLNA. In summary, our data suggest that FLNA is crucially involved in balancing stem cell characteristics and invasive properties in human seminoma cells and possibly human testicular germ cells.
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http://dx.doi.org/10.3390/cells9122563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761120PMC
November 2020

Efficacy of HDAC Inhibitors Belinostat and Panobinostat against Cisplatin-Sensitive and Cisplatin-Resistant Testicular Germ Cell Tumors.

Cancers (Basel) 2020 Oct 10;12(10). Epub 2020 Oct 10.

Cancer Biology and Epigenetics Group, IPO Porto Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal.

Novel treatment options are needed for testicular germ cell tumor (TGCT) patients, particularly important for those showing or developing cisplatin resistance, the major cause of cancer-related deaths. As TGCTs pathobiology is highly related to epigenetic (de)regulation, epidrugs are potentially effective therapies. Hence, we sought to explore, for the first time, the effect of the two most recently FDA-approved HDAC inhibitors (HDACis), belinostat and panobinostat, in (T)GCT cell lines including those resistant to cisplatin. In silico results were validated in 261 patient samples and differential expression of HDACs was also observed across cell lines. Belinostat and panobinostat reduced cell viability in both cisplatin-sensitive cells (NCCIT-P, 2102Ep-P, and NT2-P) and, importantly, also in matched cisplatin-resistant subclones (NCCIT-R, 2102Ep-R, and NT2-R), with IC50s in the low nanomolar range for all cell lines. Treatment of NCCIT-R with both drugs increased acetylation, induced cell cycle arrest, reduced proliferation, decreased Ki67 index, and increased p21, while increasing cell death by apoptosis, with upregulation of cleaved caspase 3. These findings support the effectiveness of HDACis for treating TGCT patients in general, including those developing cisplatin resistance. Future studies should explore them as single or combination agents.
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http://dx.doi.org/10.3390/cancers12102903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601457PMC
October 2020

The detection of isochromosome i(12p) in malignant germ cell tumours and tumours with somatic malignant transformation by the use of quantitative real-time polymerase chain reaction.

Histopathology 2021 Mar 21;78(4):593-606. Epub 2020 Nov 21.

Institute of Pathology, University Medical Centre, Göttingen, Germany.

Aims: Malignant germ cell tumours (GCTs) of the testis are rare neoplasms, but the most common solid malignancies in young men. World Health Organization guidelines divide GCTs into five types, for which numerous immunohistochemical markers allow exact histological subtyping in the majority of cases. In contrast, a germ cell origin is often hard to prove in metastatic GCTs that have developed so-called somatic malignant transformation. A high percentage, up to 89%, of GCTs are characterised by the appearance of isochromosome 12p [i(12p)]. Fluorescence in-situ hybridisation has been the most common diagnostic method for the detection of i(12p) so far, but has the disadvantages of being time-consuming, demanding, and not being a stand-alone method. The aim of the present study was to establish a quantitative real-time polymerase chain reaction assay as an independent method for detecting i(12p) and regional amplifications of the short arm of chromosome 12 by using DNA extracted from formalin-fixed paraffin-embedded tissue.

Methods And Results: A cut-off value to distinguish between the presence and absence of i(12p) was established in a control set consisting of 36 tumour-free samples. In a training set of 149 GCT samples, i(12p) was detectable in 133 tumours (89%), but not in 16 tumours (11%). In a test set containing 27 primary and metastatic GCTs, all 16 tumours with metastatic spread and/or somatic malignant transformation were successfully identified by the detection of i(12p).

Conclusion: In summary, the qPCR assay presented here can help to identify, further characterise and assign a large proportion of histologically inconclusive malignancies to a GCT origin.
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http://dx.doi.org/10.1111/his.14258DOI Listing
March 2021

Evaluation of Chemotherapeutic Drugs for Treatment of (Cisplatin-Resistant) Germ Cell Cancer Cell Lines.

Methods Mol Biol 2021 ;2195:99-111

Department of Urology, Urological Research Laboratory, Translational UroOncology, University Hospital Düsseldorf, Düsseldorf, Germany.

Cisplatin resistance still remains a major obstacle in the standard chemotherapeutic approach in late-stage and metastatic testicular germ cell cancer (GCC) patients. This multifactorial and complex phenomenon arises (concomitantly) on several levels due to impaired transport, decreased adduct formation, increased DNA-repair, decreased apoptosis, or compensating pathways. Evaluation of novel therapeutic approaches and pharmacological inhibitors still remains necessary to treat cisplatin-resistant GCCs. In this chapter, we present in vitro techniques to measure cytotoxic impacts of chemotherapeutic drugs on GCC cell lines. Specifically, we will discuss the measurement of relative cell viability by XTT assay, as well as cell cycle distribution and apoptosis assay by Nicoletti- and Annexin V/PI apoptosis assay with subsequent flow cytometry, respectively, to evaluate the effects of cytotoxic treatment in GCC cell lines.
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http://dx.doi.org/10.1007/978-1-0716-0860-9_8DOI Listing
March 2021

Cultivation of Testicular Germ Cell Cancer Cell Lines and Establishment of Gene-Edited Subclones Using CRISPR/Cas9.

Methods Mol Biol 2021 ;2195:85-97

Department of Developmental Pathology, Institute of Pathology, University Hospital Bonn, Bonn, Germany.

Type II testicular germ cell tumors (GCTs) can be classified as seminoma or embryonal carcinoma. Both subtypes present distinct cellular morphologies and characteristics. Seminomas closely resemble primordial germ cells (PGCs) with respect to their transcriptome and epigenetic signature (DNA hypomethylation). They express the pluripotency markers LIN28, NANOG, and OCT3/4 and the PGC markers SOX17, PRDM1, TFAP2C, DMRT1, and cKIT. Embryonal carcinomas show increased levels of DNA methylation (hypermethylation). They also express the pluripotency markers LIN28, NANOG, and OCT3/4, but additionally DNMT3B and SOX2. In contrast to seminomas, these tumors are pluripotent to totipotent and thus able to differentiate into cells of all three germ layers (teratoma) and extraembryonic tissues (yolk-sac tumor, choriocarcinoma). This protocol summarizes the essential techniques for standard cultivation of seminoma (TCam-2), embryonal carcinoma (NCCIT, NT2/D1, 2102EP), and choriocarcinoma (JEG-3, JAR) cell lines, as well as the methods to establish gene-edited subclones using the CRISPR/Cas9 system.
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http://dx.doi.org/10.1007/978-1-0716-0860-9_7DOI Listing
March 2021

Three-Dimensional Cultivation of Germ Cell Cancer Cell Lines as Hanging Drops.

Methods Mol Biol 2021 ;2195:77-83

Department of Urology, Urological Research Laboratory, Translational UroOncology, University Hospital Düsseldorf, Düsseldorf, Germany.

The hanging drop cell culture technique allows to study three-dimensional growth and differentiation of cell aggregates, that is, embryonic stem cells. Compared to standard two-dimensional monolayer cell cultivation, hanging drops allow for a better visualization and understanding of the developmental processes in vitro. Hanging drop cultivation can also be used to study biology of cancer cells three-dimensionally in vitro. This method can serve as an intermediate between the two-dimensional monolayer cell culture and in vivo models, which can be simply established in laboratories exhibiting minimum requirements of cell culture equipment. In this chapter, we describe the three-dimensional cultivation of germ cell cancer cell lines in hanging drops.
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http://dx.doi.org/10.1007/978-1-0716-0860-9_6DOI Listing
March 2021

Knockdown of Myoferlin Suppresses Migration and Invasion in Clear-Cell Renal-Cell Carcinoma.

Anticancer Res 2020 Jun;40(6):3119-3128

Department of Urology, University Hospital Bonn, Bonn, Germany.

Background/aim: Myoferlin (MYOF) has emerged as an oncogenic protein in various human cancer types. This study was conducted to investigate comprehensively the expression and functional properties of MYOF in clear-cell renal-cell carcinoma (ccRCC) with respect to its value as diagnostic biomarker and therapeutic target.

Materials And Methods: mRNA and protein expression of MYOF were assessed by quantitative polymerase chain reaction and immunohistochemistry. siRNA-mediated knockdown of MYOF was performed in the RCC cell line ACHN followed by proliferation, migration and invasion assays.

Results: MYOF mRNA and protein expression were significantly up-regulated in ccRCC. Higher mRNA levels were measured in advanced tumors. MYOF protein expression was increased in tumors with higher histological grades, and those with positive lymph node and surgical margin status. MYOF knockdown led to reduction of migration and invasion in ACHN cells, whereas expression of angiogenesis-associated genes tyrosine-protein kinase receptor-2 (TIE2), angiopoietin 2 (ANG2) and caveolin-1 (CAV1) was up-regulated following knockdown.

Conclusion: MYOF may serve as a diagnostic biomarker of tumor progression and a potential therapeutic target in ccRCC.
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http://dx.doi.org/10.21873/anticanres.14293DOI Listing
June 2020

Targeting EpCAM by a Bispecific Trifunctional Antibody Exerts Profound Cytotoxic Efficacy in Germ Cell Tumor Cell Lines.

Cancers (Basel) 2020 May 19;12(5). Epub 2020 May 19.

Department of Pediatric Hematology and Oncology, University Hospital Bonn, University of Bonn, 53127 Bonn, Germany.

Outcome in high-risk patients with refractory or relapsed germ cell tumours (GCT) remains poor. Novel strategies enhancing therapeutic efficacy whilst limiting therapeutic burden are warranted, yet immunotherapy approaches geared towards activating endogenous antitumor responses have not been successful thus far. Redirection of cytotoxic effector cells by bispecific antibodies represents a promising approach in this setting. We demonstrate that the Epithelial Cell Adhesion Molecule (EpCAM) is broadly expressed in GCT cell lines of different histologic origin including seminoma, choriocarcinoma (CHC), and embryonal carcinoma (EC). In these GCT lines of variable EpCAM surface expression, targeting T cells by the prototypic bispecific EpCAM/CD3-antibody (bAb) Catumaxomab together with natural killer (NK) cell engagement via the Fc domain promotes profound cytotoxicity across a broad range of antibody dilutions. In contrast, tumor cell lysis mediated by either immune cell subset alone is influenced by surface density of the target antigen. In the CHC line JAR, NK cell-dependent cytotoxicity dominates, which may be attributed to differential surface expression of immunomodulatory proteins such as MHC-I, CD24, and Fas receptors on CHC and EC. In view of redirecting T cell therapy mediated by bispecific antibodies, such differences in GCT immunophenotype potentially favoring immune escape are worth further investigation.
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http://dx.doi.org/10.3390/cancers12051279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281168PMC
May 2020

The contrasting roles of Dysferlin during tumor progression in renal cell carcinoma.

Urol Oncol 2020 08 17;38(8):687.e1-687.e11. Epub 2020 May 17.

Department of Urology, University Hospital Bonn, Bonn, Germany.

Background: The vesicle fusion protein Dysferlin (DYSF) is mainly known as a membrane repair protein in muscle cells. Mutations of DYSF lead to muscular dystrophies and cardiomyopathies. In contrast to other members of the Ferlin protein family, few is known about its role in cancer. Our study was designed to investigate the expression and functional properties of DYSF in ccRCC and its association with clinicopathological parameters and survival.

Material And Methods: TCGA cohort: mRNA expression data of DYSF were extracted from TCGA for patients with ccRCC (n = 603; ccRCC n = 522, benign n = 81). Study cohort: mRNA expression of DYSF in ccRCC was determined using qPCR (n = 126; ccRCC n = 82, benign n = 44). Immunohistochemical staining against DYSF was performed on tissue microarrays to validate protein expression (n = 172; ccRCC n = 142, benign n = 30). Correlations between mRNA/protein expression and clinicopathological data were statistically tested. Following siRNA-mediated knockdown of DYSF in ccRCC cell line ACHN, cell migration, invasion and proliferation were investigated.

Results: Both DYSF mRNA and protein expression are significantly up-regulated in ccRCC tissue. DYSF mRNA expression decreased during tumor progression: lower expression levels were measured in higher stage/grade and metastatic ccRCC with independent prognostic significance for overall and cancer-specific survival. In contrast, protein expression correlated positively with pathological parameters. Overexpression showed tendency toward poor survival. Accordingly, knockdown of DYSF suppressed migration and invasion of ccRCC cells.

Conclusion: DYSF mRNA and protein expression are opposingly involved in tumor progression of ccRCC. DYSF could be used as a prognostic biomarker to predict survival of patients with ccRCC.
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http://dx.doi.org/10.1016/j.urolonc.2020.04.021DOI Listing
August 2020

CDK4/6 inhibition presents as a therapeutic option for paediatric and adult germ cell tumours and induces cell cycle arrest and apoptosis via canonical and non-canonical mechanisms.

Br J Cancer 2020 08 18;123(3):378-391. Epub 2020 May 18.

Department of Urology, Urological Research Lab, Translational UroOncology, University Hospital Düsseldorf, Düsseldorf, Germany.

Background: Germ cell tumours (GCTs) are the most common solid malignancies in young men. Although high cure rates can be achieved, metastases, resistance to cisplatin-based therapy and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In this study, we analysed the potential of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors palbociclib and ribociclib (PaRi) as molecular drugs to treat cisplatin-resistant and -sensitive paediatric and adult GCTs.

Methods: Ten GCT cell lines, including cisplatin-resistant subclones and non-malignant controls, were treated with PaRi and screened for changes in viability (triphenyl tetrazolium chloride (XTT) assay), apoptosis rates (flow cytometry, caspase assay), the cell cycle (flow cytometry), the transcriptome (RNA-sequencing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and on protein level (western blot). Expression profiling was performed on paediatric and adult GCT tissues (expression microarrays, qRT-PCR, immunohistochemistry, 'The Cancer Genome Atlas' database).

Results: We demonstrate that adult GCTs highly express CDK4, while paediatric GCTs strongly express CDK6 instead. Thus, both GCT types are potentially treatable by PaRi. GCTs presented as highly sensitive towards PaRi, which caused a decrease in viability, cell cycle arrest and apoptosis. Although GCTs mainly arrested in the G1/G0 phase, some embryonal carcinoma cell lines were able to bypass the G1/S checkpoint and progressed to the G2/M phase. We found that upregulation of CDK3 and downregulation of many mitosis regulation factors, like the HAUS genes, might be responsible for bypassing the G1/S checkpoint and termination of mitosis, respectively. We postulate that GCT cells do not tolerate these alterations in the cell cycle and eventually induce apoptosis.

Conclusion: Our study highlights PaRi as therapeutic options for cisplatin-resistant and -sensitive paediatric and adult GCTs.
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http://dx.doi.org/10.1038/s41416-020-0891-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403155PMC
August 2020

Regulates Transcription and the Epigenetic Landscape via POLE and DMAP1 while Deficiency or Pharmacological Inhibition Sensitizes Germ Cell Tumor Cells to ATR Inhibition.

Cancers (Basel) 2020 Apr 7;12(4). Epub 2020 Apr 7.

Department of Urology, Urological Research Lab, Translational UroOncology, University Hospital Düsseldorf, 40225 Düsseldorf, Germany.

Germ cell tumors (GCTs) are the most common solid malignancies found in young men. Although they generally have high cure rates, metastases, resistance to cisplatin-based therapy, and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In a previous study, we identified downregulation of the chromatin-remodeling SWI/SNF complex member ARID1A as a key event in the mode of action of the histone deacetylase inhibitor romidepsin. Additionally, the loss-of-function mutations re-sensitize different tumor types to various drugs, like EZH2-, PARP-, HDAC-, HSP90- or ATR-inhibitors. Thus, ARID1A presents as a promising target for synthetic lethality and combination therapy. In this study, we deciphered the molecular function of ARID1A and screened for the potential of two pharmacological ARID1A inhibitors as a new therapeutic strategy to treat GCTs. By CRISPR/Cas9, we generated -deficient GCT cells and demonstrate by mass spectrometry that is putatively involved in regulating transcription, DNA repair and the epigenetic landscape via DNA Polymerase POLE and the DNA methyltransferase 1-associated protein DMAP1. Additionally, deficiency or pharmacological inhibition increased the efficacy of romidepsin and considerably sensitized GCT cells, including cisplatin-resistant subclones, towards ATR inhibition. Thus, targeting ARID1A in combination with romidepsin and ATR inhibitors presents as a new putative option to treat GCTs.
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http://dx.doi.org/10.3390/cancers12040905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226530PMC
April 2020

Unique and redundant roles of SOX2 and SOX17 in regulating the germ cell tumor fate.

Int J Cancer 2020 03 1;146(6):1592-1605. Epub 2019 Nov 1.

Department of Developmental Pathology, Institute of Pathology, University of Bonn Medical School, Bonn, Germany.

Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma-like TCam-2 cells to SOX17 in somatic cells and SOX2 in EC-like 2102EP cells. In seminoma-like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17-binding sites overlap in seminoma-like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma-like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC-like cells SOX2 regulates pluripotency-associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.
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http://dx.doi.org/10.1002/ijc.32714DOI Listing
March 2020

Management, Treatment, and Molecular Background of the Growing Teratoma Syndrome.

Urol Clin North Am 2019 Aug 29;46(3):419-427. Epub 2019 May 29.

Department of Urology, University of Duesseldorf, Medical Faculty, Heinrich-Heine-University, Moorenstr. 5, Duesseldorf 40225, Germany.

Growing teratoma syndrome (GTS) is a rare clinical phenomenon in patients with nonseminomatous germ cell cancer defined by growing metastatic mass during ongoing or directly after completed chemotherapy with timely decreasing tumor markers and postpubertal teratoma exclusively after resection. GTS was first described in 1982, and few reports have been published. The limited number of studies and the resulting lack of exact knowledge about development, differentiation, and treatment of GTS leaves several clinical problems regarding treatment and follow-up unsolved. This review provides an overview of clinical diagnosis and disease management and an approach to explain the molecular development of GTS.
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http://dx.doi.org/10.1016/j.ucl.2019.04.008DOI Listing
August 2019

TCam-2 Cells Deficient for SOX2 and FOXA2 Are Blocked in Differentiation and Maintain a Seminoma-Like Cell Fate In Vivo.

Cancers (Basel) 2019 May 25;11(5). Epub 2019 May 25.

Department of Developmental Pathology, Institute of Pathology, Bonn University Medical School, 53127 Bonn, Germany.

Testicular germ cell tumors (GCTs) are very common in young men and can be stratified into seminomas and non-seminomas. While seminomas share a similar gene expression and epigenetic profile with primordial germ cells, the stem cell population of the non-seminomas, the embryonal carcinoma (EC), resembles malignant embryonic stem cells. Thus, ECs are able to differentiate into cells of all three germ layers (teratomas) and even extra-embryonic-tissue-like cells (yolk-sac tumor, choriocarcinoma). In the last years, we demonstrated that the cellular microenvironment considerably influences the plasticity of seminomas (TCam-2 cells). Upon a microenvironment-triggered inhibition of the BMP signaling pathway in vivo (murine flank or brain), seminomatous TCam-2 cells reprogram to an EC-like cell fate. We identified SOX2 as a key factor activated upon BMP inhibition mediating the reprogramming process by regulating pluripotency, reprogramming and epigenetic factors. Indeed, CRISPR/Cas9 SOX2-deleted TCam-2 cells were able to maintain a seminoma-cell fate in vivo for about six weeks, but after six weeks in vivo still small sub-populations initiated differentiation. Closer analyses of these differentiated clusters suggested that the pioneer factor FOXA2 might be the driving force behind this induction of differentiation, since many FOXA2 interacting genes and differentiation factors like , , , , , , , and were upregulated. In this study, we generated TCam-2 cells double-deficient for and using the CRISPR/Cas9 technique and xenografted those cells into the flank of nude mice. Upon loss of SOX2 and FOXA2, TCam-2 maintained a seminoma cell fate for at least twelve weeks, demonstrating that both factors are key players in the reprogramming to an EC-like cell fate. Therefore, our study adds an important piece to the puzzle of GCT development and plasticity, providing interesting insights in what can be expected in a patient, when GCT cells are confronted with different microenvironments.
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http://dx.doi.org/10.3390/cancers11050728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562827PMC
May 2019

Epigenetic drugs and their molecular targets in testicular germ cell tumours.

Nat Rev Urol 2019 04;16(4):245-259

Institute of Pathology, Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany.

Current treatment regimens for type II testicular germ cell tumours (TGCTs) achieve cure rates of ≥95%; however, 1-5% of TGCTs develop resistance to standard platinum-based chemotherapy. Patients with recurrent TGCT typically receive high-dose chemotherapy, but this treatment results in severe adverse effects and cytotoxicity. Thus, alternative treatment options should be considered to improve patient well-being and quality of life. Epigenetic drugs could be feasible options for TGCT treatment. Several compounds have already been tested in TGCT cell lines and xenograft models with promising results. These compounds include DNA demethylating agents (such as SGI-110), histone demethylase inhibitors (such as the lysine-specific histone demethylase 1A (LSD1) inhibitor CBB3001), histone deacetylase (HDAC) inhibitors (such as romidepsin) and bromodomain inhibitors (such as JQ1). Despite the diversity in their molecular effects, most epigenetic compounds show strong overlap in their genetic response. The use of epigenetic drugs in TGCTs triggers a cellular stress response, induction of differentiation and downregulation of genes associated with pluripotency, leading to growth arrest and apoptosis. Additive effects are seen using a combination of JQ1 and romidepsin. The availability of dual drugs (such as LSD1-HDAC1 hybrid inhibitors) could additionally take advantage of drug synergy effects. Thus, epigenetic drugs are novel tools that could be combined with standard therapy approaches to improve treatment of TGCTs.
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http://dx.doi.org/10.1038/s41585-019-0154-xDOI Listing
April 2019

Deciphering the molecular effects of romidepsin on germ cell tumours: DHRS2 is involved in cell cycle arrest but not apoptosis or induction of romidepsin effectors.

J Cell Mol Med 2019 01 20;23(1):670-679. Epub 2018 Nov 20.

Department of Developmental Pathology, Institute of Pathology, University Medical School Bonn, Bonn, Germany.

Testicular germ cell tumours (GCTs) mostly affect young men at age 17-40. Although high cure rates can be achieved by orchiectomy and chemotherapy, GCTs can still be a lethal threat to young patients with metastases or therapy resistance. Thus, alternative treatment options are needed. Based on studies utilising GCT cell lines, the histone deacetylase inhibitor romidepsin is a promising therapeutic option, showing high toxicity at very low doses towards cisplatin-resistant GCT cells, but not fibroblasts or Sertoli cells. In this study, we extended our analysis of the molecular effects of romidepsin to deepen our understanding of the underlying mechanisms. Patients will benefit from these analyses, since detailed knowledge of the romidepsin effects allows for a better risk and side-effect assessment. We screened for changes in histone acetylation of specific lysine residues and analysed changes in the DNA methylation landscape after romidepsin treatment of the GCT cell lines TCam-2, 2102EP, NCCIT and JAR, while human fibroblasts were used as controls. In addition, we focused on the role of the dehydrogenase/reductase DHRS2, which was strongly up-regulated in romidepsin treated cells, by generating DHRS2-deficient TCam-2 cells using CRISPR/Cas9 gene editing. We show that DHRS2 is dispensable for up-regulation of romidepsin effectors (GADD45B, DUSP1, ZFP36, ATF3, FOS, CDKN1A, ID2) but contributes to induction of cell cycle arrest. Finally, we show that a combinatory treatment of romidepsin plus the gluccocorticoid dexamethasone further boosts expression of the romidepsin effectors and reduces viability of GCT cells more strongly than under single agent treatment. Thus, romidepsin and dexamethasone might represent a new combinatorial approach for treatment of GCT.
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http://dx.doi.org/10.1111/jcmm.13971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307807PMC
January 2019

Brentuximab vedotin exerts profound antiproliferative and pro-apoptotic efficacy in CD30-positive as well as cocultured CD30-negative germ cell tumour cell lines.

J Cell Mol Med 2018 01 22;22(1):568-575. Epub 2017 Sep 22.

Department of Paediatric Haematology and Oncology, University Children's Hospital Bonn, University of Bonn Medical School, Bonn, Germany.

Prognosis in patients suffering from high-risk, refractory and relapsed germ cell tumours (GCT) often comprising of CD30-positive embryonal carcinoma (EC) components remains poor. Thus, novel treatment strategies are warranted. The antibody-drug conjugate (ADC) brentuximab vedotin delivers the potent antimitotic drug monomethyl auristatin E (MMAE) to CD30-expressing tumour cells. After CD30 binding, internalization and intracellular linker cleavage cytotoxic MMAE can efflux and eradicate neighbouring CD30-negative cells. To analyse cytotoxicity and a potential bystander effect of brentuximab vedotin in GCT, we established an in vitro coculture model mimicking GCT of heterogeneous CD30 positivity and measured cell viability, proliferation and apoptosis after exposure to brentuximab vedotin and unbound MMAE by MTS- and flow cytometry-based CFSE/Hoechst assay. CD30 expression being assessed by quantitative RT-PCR and immunohistochemistry was apparent in all EC cell lines with different intensity. Brentuximab vedotin abrogates cell viability of CD30-positive GCT27 EC line exerting marked time-dependent antiproliferative and pro-apoptotic activity. CD30-negative JAR cultured alone barely responds to brentuximab vedotin, while in coculture with GCT27 brentuximab vedotin induces clear dose-dependent cytotoxicity. Cellular proliferation and cell death are significantly enhanced in CD30-negative JAR cocultured with CD30-positive GCT27 compared to JAR cultured alone in proof of substantial bystander activity of brentuximab vedotin in CD30-negative GCT. We present first evidence that in an in vitro model mimicking GCT of heterogeneous histology, brentuximab vedotin exerts potent antiproliferative and pro-apoptotic activity against both CD30-positive as well as CD30-negative GCT subsets. Our results strongly support translational efforts to evaluate clinical efficacy of brentuximab vedotin in high-risk GCT of heterogeneous CD30 positivity.
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http://dx.doi.org/10.1111/jcmm.13344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742680PMC
January 2018

MAPK Signaling and Inflammation Link Melanoma Phenotype Switching to Induction of CD73 during Immunotherapy.

Cancer Res 2017 09 26;77(17):4697-4709. Epub 2017 Jun 26.

Unit for RNA Biology, Department of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany.

Evolution of tumor cell phenotypes promotes heterogeneity and therapy resistance. Here we found that induction of CD73, the enzyme that generates immunosuppressive adenosine, is linked to melanoma phenotype switching. Activating MAPK mutations and growth factors drove CD73 expression, which marked both nascent and full activation of a mesenchymal-like melanoma cell state program. Proinflammatory cytokines like TNFα cooperated with MAPK signaling through the c-Jun/AP-1 transcription factor complex to activate CD73 transcription by binding to an intronic enhancer. In a mouse model of T-cell immunotherapy, CD73 was induced in relapse melanomas, which acquired a mesenchymal-like phenotype. We also detected CD73 upregulation in melanoma patients progressing under adoptive T-cell transfer or immune checkpoint blockade, arguing for an adaptive resistance mechanism. Our work substantiates CD73 as a target to combine with current immunotherapies, but its dynamic regulation suggests limited value of CD73 pretreatment expression as a biomarker to stratify melanoma patients. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-0395DOI Listing
September 2017

The plasticity of germ cell cancers and its dependence on the cellular microenvironment.

J Cell Mol Med 2017 08 28;21(8):1463-1467. Epub 2017 Feb 28.

Department of Developmental Pathology, Institute of Pathology, University Medical School, Bonn, Germany.

So far, the understanding of germ cell cancer (GCC) pathogenesis is based on a model, where seminomas and non-seminomas represent distinct entities although originating from a common precursor termed germ cell neoplasia in situ (GCNIS). Embryonal carcinomas (ECs), the stem cell population of the non-seminomas, is pluri- to totipotent and able to differentiate into cells of all three germ layers, giving rise to teratomas or tumours mimicking extraembryonic tissues (yolk sac tumours, choriocarcinomas). With regard to gene expression, (epi)genetics and histology, seminomas are highly similar to GCNIS and primordial germ cells, but limited in development. It remains elusive, whether this block in differentiation is controlled by cell intrinsic mechanisms or by signals from the surrounding microenvironment. Here, we reviewed the recent literature emphasizing the plasticity of GCCs, especially of seminomas. We propose that this plasticity is controlled by the microenvironment, allowing seminomas to transit into an EC or mixed non-seminoma and vice versa. We discuss several mechanisms and routes of reprogramming that might be responsible for this change in the cell fate. We finally integrate this plasticity into a new model of GCC pathogenesis, allowing for an alternative view on the dynamics of GCC development and progression.
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http://dx.doi.org/10.1111/jcmm.13082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543455PMC
August 2017

The bromodomain inhibitor JQ1 triggers growth arrest and apoptosis in testicular germ cell tumours in vitro and in vivo.

J Cell Mol Med 2017 07 27;21(7):1300-1314. Epub 2016 Dec 27.

Institute of Pathology, Department of Developmental Pathology, University Medical School, Bonn, Germany.

Type II testicular germ cell cancers (TGCT) are the most frequently diagnosed tumours in young men (20-40 years) and are classified as seminoma or non-seminoma. TGCTs are commonly treated by orchiectomy and chemo- or radiotherapy. However, a subset of metastatic non-seminomas (embryonal carcinomas) displays only incomplete remission or relapse and requires novel treatment options. Recent studies have shown effective application of the small-molecule inhibitor JQ1 in tumour therapy, which interferes with the function of 'bromodomain and extraterminal (BET)' proteins. JQ1-treated TGCT cell lines display up-regulation of genes indicative for DNA damage and cellular stress response and induce cell cycle arrest. Embryonal carcinoma (EC) cell lines, which presented as JQ1 sensitive, display down-regulation of pluripotency factors and induction of mesodermal differentiation. In contrast, seminoma-like TCam-2 cells tolerated higher JQ1 concentrations and were resistant to differentiation. ECs xenografted in vivo showed a reduction in tumour size, proliferation rate and angiogenesis in response to JQ1. Finally, the combination of JQ1 and the histone deacetylase inhibitor romidepsin allowed for lower doses and less frequent application, compared with monotherapy. Thus, we propose that JQ1 in combination with romidepsin may serve as a novel therapeutic option for (mixed) TGCTs.
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http://dx.doi.org/10.1111/jcmm.13059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487916PMC
July 2017

Re-visiting the Protamine-2 locus: deletion, but not haploinsufficiency, renders male mice infertile.

Sci Rep 2016 11 11;6:36764. Epub 2016 Nov 11.

Institute of Pathology, Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany.

Protamines are arginine-rich DNA-binding proteins that replace histones in elongating spermatids. This leads to hypercondensation of chromatin and ensures physiological sperm morphology, thereby protecting DNA integrity. In mice and humans, two protamines, protamine-1 (Prm1) and protamine-2 (Prm2) are expressed in a species-specific ratio. In humans, alterations of this PRM1/PRM2 ratio is associated with subfertility. By applying CRISPR/Cas9 mediated gene-editing in oocytes, we established Prm2-deficient mice. Surprisingly, heterozygous males remained fertile with sperm displaying normal head morphology and motility. In Prm2-deficient sperm, however, DNA-hypercondensation and acrosome formation was severely impaired. Further, the sperm displayed severe membrane defects resulting in immotility. Thus, lack of Prm2 leads not only to impaired histone to protamine exchange and disturbed DNA-hypercondensation, but also to severe membrane defects resulting in immotility. Interestingly, previous attempts using a regular gene-targeting approach failed to establish Prm2-deficient mice. This was due to the fact that already chimeric animals generated with Prm2 ES cells were sterile. However, the Prm2-deficient mouse lines established here clearly demonstrate that mice tolerate loss of one Prm2 allele. As such they present an ideal model for further studies on protamine function and chromatin organization in murine sperm.
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http://dx.doi.org/10.1038/srep36764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5105070PMC
November 2016

Xenografting of Cancer Cell Lines for In Vivo Screening of the Therapeutic Potential of HDAC Inhibitors.

Methods Mol Biol 2017 ;1510:211-215

Department of Developmental Pathology, Institute of Pathology, University Medical School Bonn, Sigmund-Freud-Strasse 25, 53127, Bonn, Germany.

Histone deacetylase inhibitor application is lethal to many cancer types. To screen for the therapeutic potential of HDIs it is necessary to analyze their ability to target and kill cancer cells in vivo. Here, we describe the xenografting of (germ cell) cancer cell lines into the flank of nude mice and the subsequent intravenous application of HDIs.
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http://dx.doi.org/10.1007/978-1-4939-6527-4_15DOI Listing
January 2018

A signaling cascade including ARID1A, GADD45B and DUSP1 induces apoptosis and affects the cell cycle of germ cell cancers after romidepsin treatment.

Oncotarget 2016 Nov;7(46):74931-74946

Institute of Pathology, Department of Developmental Pathology, University Medical School, Bonn, Germany.

In Western countries, the incidence of testicular germ cell cancers (GCC) is steadily rising over the last decades. Mostly, men between 20 and 40 years of age are affected. In general, patients suffering from GCCs are treated by orchiectomy and radio- or chemotherapy. Due to resistance mechanisms, intolerance to the therapy or denial of chemo- / radiotherapy by the patients, GCCs are still a lethal threat, highlighting the need for alternative treatment strategies.In this study, we revealed that germ cell cancer cell lines are highly sensitive to the histone deacetylase inhibitor romidepsin in vitro and in vivo, highlighting romidepsin as a potential therapeutic option for GCC patients.Romidepsin-mediated inhibition of histone deacetylases led to disturbances of the chromatin landscape. This resulted in locus-specific histone-hyper- or hypoacetylation. We found that hypoacetylation at the ARID1A promotor caused repression of the SWI/SNF-complex member ARID1A. In consequence, this resulted in upregulation of the stress-sensors and apoptosis-regulators GADD45B, DUSP1 and CDKN1A. RNAi-driven knock down of ARID1A mimicked in parts the effects of romidepsin, while CRISPR/Cas9-mediated deletion of GADD45B attenuated the romidepsin-provoked induction of apoptosis and cell cycle alterations.We propose a signaling cascade involving ARID1A, GADD45B and DUSP1 as mediators of the romidepsin effects in GCC cells.
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http://dx.doi.org/10.18632/oncotarget.11647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342713PMC
November 2016

Elucidating human male germ cell development by studying germ cell cancer.

Reproduction 2016 10;152(4):R101-13

Department of Developmental PathologyInstitute of Pathology, University of Bonn Medical School, Bonn, Germany

Human germ cell development is regulated in a spatio-temporal manner by complex regulatory networks. Here, we summarize results obtained in germ cell tumors and respective cell lines and try to pinpoint similarities to normal germ cell development. This comparison allows speculating about the critical and error-prone mechanisms, which when disturbed, lead to the development of germ cell tumors. Short after specification, primordial germ cells express markers of pluripotency, which, in humans, persists up to the stage of fetal/infantile spermatogonia. Aside from the rare spermatocytic tumors, virtually all seminomas and embryonal carcinomas express markers of pluripotency and show signs of pluripotency or totipotency. Therefore, it appears that proper handling of the pluripotency program appears to be the most critical step in germ cell development in terms of tumor biology. Furthermore, data from mice reveal that germline cells display an epigenetic signature, which is highly similar to pluripotent cells. This signature (poised histone code, DNA hypomethylation) is required for the rapid induction of toti- and pluripotency upon fertilization. We propose that adult spermatogonial cells, when exposed to endocrine disruptors or epigenetic active substances, are prone to reinitiate the pluripotency program, giving rise to a germ cell tumor. The fact that pluripotent cells can be derived from adult murine and human testicular cells further corroborates this idea.
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http://dx.doi.org/10.1530/REP-16-0114DOI Listing
October 2016
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