Publications by authors named "Heike Peterziel"

20 Publications

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A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors.

Cancers (Basel) 2021 Jan 10;13(2). Epub 2021 Jan 10.

Division of Signal Transduction and Growth Control, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.

Glioblastomas (GBM) are the most aggressive tumors affecting the central nervous system in adults, causing death within, on average, 15 months after diagnosis. Immunocompetent in-vivo models that closely mirror human GBM are urgently needed for deciphering glioma biology and for the development of effective treatment options. The murine GBM cell lines currently available for engraftment in immunocompetent mice are not only exiguous but also inadequate in representing prominent characteristics of human GBM such as infiltrative behavior, necrotic areas, and pronounced tumor heterogeneity. Therefore, we generated a set of glioblastoma cell lines by repeated in vivo passaging of cells isolated from a neural stem cell-specific double-knockout genetic mouse brain tumor model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts, they formed high-grade gliomas that faithfully recapitulated the histopathological features, invasiveness and immune cell infiltration characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioblastoma pathomechanism and to test novel treatments in an intact immune microenvironment.
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http://dx.doi.org/10.3390/cancers13020230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827614PMC
January 2021

Rapid In Vivo Validation of HDAC Inhibitor-Based Treatments in Neuroblastoma Zebrafish Xenografts.

Pharmaceuticals (Basel) 2020 Oct 27;13(11). Epub 2020 Oct 27.

Hopp Children's Cancer Center Heidelberg (KiTZ), 69120 Heidelberg, Germany.

The survival rate among children with relapsed neuroblastomas continues to be poor, and thus new therapeutic approaches identified by reliable preclinical drug testing models are urgently needed. Zebrafish are a powerful vertebrate model in preclinical cancer research. Here, we describe a zebrafish neuroblastoma yolk sac model to evaluate efficacy and toxicity of histone deacetylase (HDAC) inhibitor treatments. Larvae were engrafted with fluorescently labeled, genetically diverse, established cell lines and short-term cultures of patient-derived primary cells. Engrafted tumors progressed locally and disseminated remotely in an intact environment. Combination treatments involving the standard chemotherapy doxorubicin and HDAC inhibitors substantially reduced tumor volume, induced tumor cell death, and inhibited tumor cell dissemination to the tail region. Hence, this model allows for fast, cost-efficient, and reliable in vivo evaluation of toxicity and response of the primary and metastatic tumor sites to drug combinations.
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http://dx.doi.org/10.3390/ph13110345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692187PMC
October 2020

Modeling glioblastoma invasion using human brain organoids and single-cell transcriptomics.

Neuro Oncol 2020 08;22(8):1138-1149

Digital Health Center, Berlin Institute of Health and Charité, Berlin, Germany.

Background: Glioblastoma (GBM) consists of devastating neoplasms with high invasive capacity, which have been difficult to study in vitro in a human-derived model system. Therapeutic progress is also limited by cellular heterogeneity within and between tumors, among other factors such as therapy resistance. To address these challenges, we present an experimental model using human cerebral organoids as a scaffold for patient-derived GBM cell invasion.

Methods: This study combined tissue clearing and confocal microscopy with single-cell RNA sequencing of GBM cells before and after co-culture with organoid cells.

Results: We show that tumor cells within organoids extend a network of long microtubes, recapitulating the in vivo behavior of GBM. Transcriptional changes implicated in the invasion process are coherent across patient samples, indicating that GBM cells reactively upregulate genes required for their dispersion. Potential interactions between GBM and organoid cells identified by an in silico receptor-ligand pairing screen suggest functional therapeutic targets.

Conclusions: Taken together, our model has proven useful for studying GBM invasion and transcriptional heterogeneity in vitro, with applications for both pharmacological screens and patient-specific treatment selection on a time scale amenable to clinical practice.
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http://dx.doi.org/10.1093/neuonc/noaa091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594554PMC
August 2020

Podoplanin Positive Myeloid Cells Promote Glioma Development by Immune Suppression.

Front Oncol 2019 26;9:187. Epub 2019 Mar 26.

Division of Signal Transduction and Growth Control, DKFZ/ZMBH Alliance, Heidelberg, Germany.

The dynamic and interactive tumor microenvironment is conceived as a considerable parameter in tumor development and therapy response. Implementing this knowledge in the development of future cancer treatments could provide novel options in the combat of highly aggressive and difficult-to-treat tumors such as gliomas. One compartment of the tumor microenvironment that has gained growing interest is the immune system. As endogenous defense machinery the immune system has the capacity to fight against cancer cells. This, however, is frequently circumvented by tumor cells engaging immune-regulatory mechanisms that disable tumor-directed immune responses. Thus, in order to unlock the immune system against cancer cells, it is crucial to characterize in great detail individual tumor-associated immune cell subpopulations and dissect whether and how they influence immune evasion. In this study we investigated the function of a tumor-associated myeloid cell subpopulation characterized by podoplanin expression on the development of high-grade glioma tumors. Here, we show that the deletion of podoplanin in myeloid cells results in increased (CD8) T-cell infiltrates and significantly prolonged survival in an orthotopic transplantation model. co-cultivation experiments indicate a podoplanin-dependent transcriptional regulation of arginase-1, a well-known player in myeloid cell-mediated immune suppression. These findings identify podoplanin positive myeloid cells as one novel mediator of the glioma-induced immune suppression. Thus, the targeted ablation of podoplanin positive myeloid cells could be included in combinatorial cancer therapies to enhance immune-mediated tumor elimination.
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http://dx.doi.org/10.3389/fonc.2019.00187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443903PMC
March 2019

Intratumoral platelet aggregate formation in a murine preclinical glioma model depends on podoplanin expression on tumor cells.

Blood Adv 2019 04;3(7):1092-1102

Division of Signal Transduction and Growth Control, German Cancer Research Center (DKFZ)-Center for Molecular Biology of the University of Heidelberg (ZMBH) Alliance, DKFZ, Heidelberg, Germany.

Binding of the sialomucin-like transmembrane glycoprotein podoplanin (PDPN) to the platelet receptor C-type lectin-like receptor 2 induces platelet activation and aggregation. In human high-grade gliomas, PDPN is highly expressed both in tumor cells and in tumor-associated astrocytes. In glioma patients, high expression of PDPN is associated with worse prognosis and has been shown to correlate with intratumoral platelet aggregation and an increased risk of venous thromboembolism (VTE). To functionally assess the role of PDPN in platelet aggregation in vivo, we established a syngeneic orthotopic murine glioma model in C57/Bl6 mice, based on transplantation of - and -deficient neural stem cells. This model is characterized by the presence of intratumoral platelet aggregates and by the upregulation of PDPN both in glioma cells and in astrocytes, reflecting the characteristics of human gliomas. Deletion of PDPN either in tumor cells or in astrocytes resulted in glioma formation with similar penetrance and grade compared with control mice. Importantly, only the lack of PDPN in tumor cells, but not in astrocytes, caused a significant reduction in intratumoral platelet aggregates, whereas in vitro, both cell types have similar platelet aggregation-inducing capacities. Our results demonstrate a causative link between PDPN and platelet aggregation in gliomas and pinpoint the tumor cells as the major players in PDPN-induced platelet aggregation. Our data indicate that blocking PDPN specifically on tumor cells could represent a novel strategy to prevent platelet aggregation and thereby reduce the risk of VTE in glioma patients.
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http://dx.doi.org/10.1182/bloodadvances.2018015966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457221PMC
April 2019

Podoplanin expression is a prognostic biomarker but may be dispensable for the malignancy of glioblastoma.

Neuro Oncol 2019 02;21(3):326-336

Division of Signal Transduction and Growth Control, DKFZ/ZMBH Alliance, Heidelberg, Germany.

Background: Treatment options of glioblastoma, the most aggressive primary brain tumor with frequent relapses and high mortality, are still very limited, urgently calling for novel therapeutic targets. Expression of the glycoprotein podoplanin correlates with poor prognosis in various cancer entities, including glioblastoma. Furthermore, podoplanin has been associated with tumor cell migration and proliferation in vitro; however, experimental data on its function in gliomagenesis in vivo are still missing. Hence, we have functionally investigated the impact of podoplanin on glioblastoma in a preclinical mouse model to evaluate its potential as a therapeutic target.

Methods: Fluorescence activated cell sorting, genome-wide expression analysis, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated deletion of podoplanin in patient-derived human glioblastoma cells were combined with organotypic brain slice cultures and intracranial injections into mice.

Results: We defined a malignant gene signature in tumor cells with high podoplanin expression. The increase and/or maintenance of high podoplanin expression in serial transplantations and in podoplaninlow-sorted glioblastoma cells during outgrowth indicated the association of high podoplanin expression and poor outcome. Unexpectedly, similar rates of proliferation, apoptosis, angiogenesis, and invasion were observed in control and podoplanin-deleted tumors. Accordingly, neither tumor growth nor survival was affected upon podoplanin loss.

Conclusion: We report that tumor progression occurs independently of podoplanin. Thus, in contrast to previous suggestions, blocking of podoplanin does not represent a promising therapeutic approach. However, as podoplanin is associated with tumor aggressiveness and progression, we propose the cell surface protein as a biomarker for poor prognosis.
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http://dx.doi.org/10.1093/neuonc/noy184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380419PMC
February 2019

Dual role of HDAC10 in lysosomal exocytosis and DNA repair promotes neuroblastoma chemoresistance.

Sci Rep 2018 07 3;8(1):10039. Epub 2018 Jul 3.

Preclinical Program, Hopp Children's Cancer Center at NCT Heidelberg (KiTZ), Heidelberg, Germany.

Drug resistance is a leading cause for treatment failure in many cancers, including neuroblastoma, the most common solid extracranial childhood malignancy. Previous studies from our lab indicate that histone deacetylase 10 (HDAC10) is important for the homeostasis of lysosomes, i.e. acidic vesicular organelles involved in the degradation of various biomolecules. Here, we show that depleting or inhibiting HDAC10 results in accumulation of lysosomes in chemotherapy-resistant neuroblastoma cell lines, as well as in the intracellular accumulation of the weakly basic chemotherapeutic doxorubicin within lysosomes. Interference with HDAC10 does not block doxorubicin efflux from cells via P-glycoprotein inhibition, but rather via inhibition of lysosomal exocytosis. In particular, intracellular doxorubicin does not remain trapped in lysosomes but also accumulates in the nucleus, where it promotes neuroblastoma cell death. Our data suggest that lysosomal exocytosis under doxorubicin treatment is important for cell survival and that inhibition of HDAC10 further induces DNA double-strand breaks (DSBs), providing additional mechanisms that sensitize neuroblastoma cells to doxorubicin. Taken together, we demonstrate that HDAC10 inhibition in combination with doxorubicin kills neuroblastoma, but not non-malignant cells, both by impeding drug efflux and enhancing DNA damage, providing a novel opportunity to target chemotherapy resistance.
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http://dx.doi.org/10.1038/s41598-018-28265-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030077PMC
July 2018

A kinome-wide RNAi screen identifies ALK as a target to sensitize neuroblastoma cells for HDAC8-inhibitor treatment.

Cell Death Differ 2018 12 7;25(12):2053-2070. Epub 2018 Mar 7.

Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany.

The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here, we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3-6 µM PCI-34051 or 10 µM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05 µM (ALK-amplified) to 0.8 µM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n = 88) and the German Neuroblastoma Trial cohort (n = 649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.
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http://dx.doi.org/10.1038/s41418-018-0080-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261943PMC
December 2018

An advanced glioma cell invasion assay based on organotypic brain slice cultures.

BMC Cancer 2018 01 30;18(1):103. Epub 2018 Jan 30.

Division of Signal Transduction and Growth Control, DKFZ/ZMBH Alliance, Heidelberg, Germany.

Background: The poor prognosis for glioblastoma patients is caused by the diffuse infiltrative growth pattern of the tumor. Therefore, the molecular and cellular processes underlying cell migration continue to be a major focus of glioblastoma research. Emerging evidence supports the concept that the tumor microenvironment has a profound influence on the functional properties of tumor cells. Accordingly, substantial effort must be devoted to move from traditional two-dimensional migration assays to three-dimensional systems that more faithfully recapitulate the complex in vivo tumor microenvironment.

Methods: In order to mimic the tumor microenvironment of adult gliomas, we used adult organotypic brain slices as an invasion matrix for implanted, fluorescently labeled tumor spheroids. Cell invasion was imaged by confocal or epi-fluorescence microscopy and quantified by determining the average cumulative sprout length per spheroid. The tumor microenvironment was manipulated by treatment of the slice with small molecule inhibitors or using different genetically engineered mouse models as donors.

Results: Both epi-fluorescence and confocal microscopy were applied to precisely quantify cell invasion in this ex vivo approach. Usage of a red-emitting membrane dye in addition to tissue clearing drastically improved epi-fluorescence imaging. Preparation of brain slices from of a genetically engineered mouse with a loss of a specific cell surface protein resulted in significantly impaired tumor cell invasion. Furthermore, jasplakinolide treatment of either tumor cells or brain slice significantly reduced tumor cell invasion.

Conclusion: We present an optimized invasion assay that closely reflects in vivo invasion by the implantation of glioma cells into organotypic adult brain slice cultures with a preserved cytoarchitecture. The diversity of applications including manipulation of the tumor cells as well as the microenvironment, permits the investigation of rate limiting factors of cell migration in a reliable context. This model will be a valuable tool for the discovery of the molecular mechanisms underlying glioma cell invasion and, ultimately, the development of novel therapeutic strategies.
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http://dx.doi.org/10.1186/s12885-018-4007-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789681PMC
January 2018

Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance.

Cell Death Dis 2017 08 24;8(8):e3013. Epub 2017 Aug 24.

Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), INF 280, D-69120 Heidelberg, Germany.

Current preclinical models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes, including autophagy. Three-dimensional (3D) growth cultures have frequently been proposed to overcome the lack of correlation between two-dimensional (2D) monolayer cell cultures and human tumors in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate shear stress, compound flux and removal of metabolites, e.g., via bioreactor systems, through which culture medium is constantly pumped at a flow rate reflecting physiological conditions. Here we show that both static 3D growth and 3D growth within a bioreactor system modulate key hallmarks of cancer cells, including proliferation and cell death as well as macroautophagy, a recycling pathway often activated by highly proliferative tumors to cope with metabolic stress. The autophagy-related gene expression profiles of 2D-grown cells are substantially different from those of 3D-grown cells and tumor tissue. Autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditions. Three-dimensional cultures depleted of the autophagy mediators BECN1, ATG5 or ATG7 or the transcription factor FOXO3, are more sensitive to cytotoxic treatment. Accordingly, combining cytotoxic treatment with compounds affecting late autophagic flux, such as chloroquine, renders the 3D-grown cells more susceptible to therapy. Altogether, 3D cultures are a valuable tool to study drug response of tumor cells, as these models more closely mimic tumor (patho-)physiology, including the upregulation of tumor relevant pathways, such as autophagy.
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http://dx.doi.org/10.1038/cddis.2017.398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596581PMC
August 2017

Derivation of Pluripotent Cells from Mouse SSCs Seems to Be Age Dependent.

Stem Cells Int 2016 9;2016:8216312. Epub 2015 Nov 9.

Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.

Here, we aimed to answer important and fundamental questions in germ cell biology with special focus on the age of the male donor cells and the possibility to generate embryonic stem cell- (ESC-) like cells. While it is believed that spermatogonial stem cells (SSCs) and truly pluripotent ESC-like cells can be isolated from adult mice, it remained unknown if the spontaneous conversion of SSCs to ESC-like cells fails at some age. Similarly, there have been differences in the literature about the duration of cultures during which ESC-like cells may appear. We demonstrate the possibility to derive ESC-like cells from SSC cultures until they reach adolescence or up to 7 weeks of age, but we point out the impossibility to derive these cells from older, mature adult mice. The inability of real adult SSCs to shift to a pluripotent state coincides with a decline in expression of the core pluripotency genes Oct4, Nanog, and Sox2 in SSCs with age. At the same time genes of the spermatogonial differentiation pathway increase. The generated ESC-like cells were similar to ESCs and express pluripotency markers. In vitro they differentiate into all three germ lineages; they form complex teratomas after transplantation in SCID mice and produce chimeric mice.
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http://dx.doi.org/10.1155/2016/8216312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655302PMC
December 2015

Neutralization of the CD95 ligand by APG101 inhibits invasion of glioma cells in vitro.

Anticancer Drugs 2015 Aug;26(7):716-27

aApogenix GmbH bDivision of Signal Transduction and Growth Control DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Glioblastoma is a disease characterized by rapid invasive tumour growth. Studies on the proapoptotic CD95/CD95L signalling pathway recently suggested a significant contribution of CD95 signalling towards the high degree of motility in glioma cells. Apogenix has developed APG101, a clinical phase II compound designed to bind and neutralize CD95L, and thus to interfere with CD95/CD95L-based signalling. APG101 has shown clinical efficacy in a controlled randomized phase II trial in patients with recurrent glioma. Because APG101 is not cytotoxic to tumour cells in vitro, we postulated that the anti-invasive function of APG101 is the main mechanism of action for this compound. Using three-dimensional spheroid invasion assays in vitro and in murine brain tissue cultures, we found that knockdown of endogenous CD95L reduced the invasive phenotype in our two glioblastoma model cell lines U87-MG and U251-MG. Invasion was restored in CD95L knockdown cells upon the addition of soluble recombinant CD95L and this effect was inhibited by APG101. We conclude that CD95L from autocrine and paracrine sources contributes towards the invasive phenotype of glioblastoma cells and that APG101 acts as a suppressor of proinvasive signalling by the CD95/CD95L pathway in glioblastoma.
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http://dx.doi.org/10.1097/CAD.0000000000000237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4484665PMC
August 2015

KIAA1797/FOCAD encodes a novel focal adhesion protein with tumour suppressor function in gliomas.

Brain 2012 Apr 16;135(Pt 4):1027-41. Epub 2012 Mar 16.

Institute of Human Genetics, Biomedical Center (BMZ), University of Bonn, 53105 Bonn, Germany.

In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.
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http://dx.doi.org/10.1093/brain/aws045DOI Listing
April 2012

Expression of podoplanin in human astrocytic brain tumors is controlled by the PI3K-AKT-AP-1 signaling pathway and promoter methylation.

Neuro Oncol 2012 Apr 6;14(4):426-39. Epub 2012 Mar 6.

Divisions of Signal Transduction and Growth Control, DKFZ/ZMBH Alliance, Heidelberg, Germany.

Recently, we found strong overexpression of the mucin-type glycoprotein podoplanin (PDPN) in human astrocytic brain tumors, specifically in primary glioblastoma multiforme (GB). In the current study, we show an inverse correlation between PDPN expression and PTEN levels in primary human GB and glioma cell lines, and we report elevated PDPN protein levels in the subventricular zone of brain tissue sections of PTEN-deficient mice. In human glioma cells lacking functional PTEN, reintroduction of wild-type PTEN, inhibition of the PTEN downstream target protein kinase B/AKT, or interference with transcription factor AP-1 function resulted in efficient downregulation of PDPN expression. In addition, we observed hypoxia-dependent PDPN transcriptional control and demonstrated that PDPN expression is subject to negative transcriptional regulation by promoter methylation in human GB and in glioma cell lines. Treatment of PTEN-negative glioma cells with demethylating agents induced expression of PDPN. Together, our findings show that increased PDPN expression in human GB is caused by loss of PTEN function and activation of the PI3K-AKT-AP-1 signaling pathway, accompanied by epigenetic regulation of PDPN promoter activity. Silencing of PDPN expression leads to reduced proliferation and migration of glioma cells, suggesting a functional role of PDPN in glioma progression and malignancy. Thus, specific targeting of PDPN expression and/or function could be a promising strategy for the treatment of patients with primary GB.
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http://dx.doi.org/10.1093/neuonc/nos055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309862PMC
April 2012

Expression and function of the kallikrein-related peptidase 6 in the human melanoma microenvironment.

J Invest Dermatol 2011 Nov 14;131(11):2281-8. Epub 2011 Jul 14.

Division of Signal Transduction and Growth Control (A100), DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany.

Cutaneous malignant melanoma is an aggressive disease of poor prognosis. Clinical and experimental studies have provided major insight into the pathogenesis of the disease, including the functional interaction between melanoma cells and surrounding keratinocytes, fibroblasts, and immune cells. Nevertheless, patients with metastasized melanoma have a very poor prognosis and are largely refractory to clinical therapies. Hence, diagnostic tools to monitor melanoma development, as well as therapeutic targets, are urgently needed. We investigated the expression pattern of the kallikrein-related peptidase 6 (KLK6) in human melanoma tissue sections throughout tumor development. Although KLK6 was not detectable in tumor cells, we found strong KLK6 protein expression in keratinocytes and stromal cells located adjacent to benign nevi, primary melanomas, and cutaneous metastatic lesions, suggesting a paracrine function of extracellular KLK6 during neoplastic transformation and malignant progression. Accordingly, recombinant Klk6 protein significantly induced melanoma cell migration and invasion accompanied by an accelerated intracellular Ca(2+) flux. We could further demonstrate that KLK6-induced intracellular Ca(2+) flux and tumor cell invasion critically depends on the protease-activated receptor 1 (PAR1). Our data provide experimental evidence that specific inhibition of the KLK6-PAR1 axis may interfere with the deleterious effect of tumor-microenvironment interaction and represent a potential option for translational melanoma research.
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http://dx.doi.org/10.1038/jid.2011.190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3193584PMC
November 2011

Genomic and expression profiling of glioblastoma stem cell-like spheroid cultures identifies novel tumor-relevant genes associated with survival.

Clin Cancer Res 2009 Nov 27;15(21):6541-50. Epub 2009 Oct 27.

Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany.

Purpose: Glioblastoma spheroid cultures are enriched in tumor stem-like cells and therefore may be more representative of the respective primary tumors than conventional monolayer cultures. We exploited the glioma spheroid culture model to find novel tumor-relevant genes.

Experimental Design: We carried out array-based comparative genomic hybridization of spheroid cultures derived from 20 glioblastomas. Microarray-based gene expression analysis was applied to determine genes with differential expression compared with normal brain tissue and to nonneoplastic brain spheroids in glioma spheroid cultures. The protein expression levels of three candidates were determined by immunohistochemistry on tissue microarrays and correlated with clinical outcome. Functional analysis of PDPN was done.

Results: Genomic changes in spheroid cultures closely resembled those detected in primary tumors of the corresponding patients. In contrast, genomic changes in serum-grown monolayer cultures established from the same patients did not match well with the respective primary tumors. Microarray-based gene expression analysis of glioblastoma spheroid cultures identified a set of novel candidate genes being upregulated or downregulated relative to normal brain. Quantitative real-time PCR analyses of 8 selected candidate genes in 20 clinical glioblastoma samples validated the microarray findings. Immunohistochemistry on tissue microarrays revealed that expression of AJAP1, EMP3, and PDPN was significantly associated with overall survival of astrocytic glioma patients. Invasive capacity and RhoA activity were decreased in PDPN-silenced spheroids.

Conclusion: We identified a set of novel candidate genes that likely play a role in glioblastoma pathogenesis and implicate AJAP1, EMP3, and PDPN as molecular markers associated with the clinical outcome of glioma patients.
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http://dx.doi.org/10.1158/1078-0432.CCR-09-0695DOI Listing
November 2009

Expression and putative functions of GDF-15, a member of the TGF-beta superfamily, in human glioma and glioblastoma cell lines.

Cancer Lett 2008 Oct 11;270(1):30-9. Epub 2008 Jun 11.

Department of Neuroanatomy and Interdisciplinary Center for Neurosciences IZN, University of Heidelberg, INF 307, Heidelberg, Germany.

Recent studies have demonstrated growth-inhibiting effects of growth differentiation factor-15 (GDF-15) on different cancer cell lines invitro and on tumor growth in vivo. Here, we present data concerning expression of GDF-15 in glioblastoma. We found low levels of GDF-15 transcripts in primary glioblastoma. Thus, GDF-15 expression might be exploited as a useful indicator for distinguishing primary from other glial derived tumors. In contrast to the documented proapoptotic and anti-tumorigenic activities of GDF-15 in several cancer cell lines, our data suggest that GDF-15 does not decrease proliferation of glioblastoma cell lines, while its effects on invasiveness are not consistent.
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http://dx.doi.org/10.1016/j.canlet.2008.04.042DOI Listing
October 2008

Specificity in the crosstalk of TGFbeta/GDNF family members is determined by distinct GFR alpha receptors.

J Neurochem 2007 Dec 22;103(6):2491-504. Epub 2007 Oct 22.

Department of Neuroanatomy, IZN, University of Heidelberg, Heidelberg, Germany.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) are neurotrophic factors for parasympathetic neurons including ciliary ganglion (CG) neurons. Recently, we have shown that survival and signaling mediated by GDNF in CG neurons essentially requires transforming growth factor beta (TGFbeta). We have provided evidence that TGFbeta regulates the availability of the glycosyl phosphatidylinositol (GPI)-anchored GDNF receptor alpha 1 (GFRalpha1) by promoting the recruitment of the receptor to the plasma membrane. We report now that in addition to GDNF, NRTN, but not persephin (PSPN) or artemin (ARTN), is able to promote survival of CG neurons. Interestingly, in contrast to GDNF, NRTN is not dependent on cooperation with TGFbeta, but efficiently promotes neuronal survival and intracellular signaling in the absence of TGFbeta. Additional treatment with TGFbeta does not further increase the NRTN response. Both NRTN and GDNF exclusively bind to and activate their cognate receptors, GFRalpha2 and GFRalpha1, respectively, as shown by the use of receptor-specific neutralizing antibodies. Immunocytochemical staining for the two receptors on the surface of CG neurons reveals that, in contrast to the effect on GFRalpha1, TGFbeta is not required for recruitment of GFRalpha2 to the plasma membrane. Moreover, binding of radioactively labeled GDNF but not NRTN is increased upon treatment of CG neurons with TGFbeta. Disruption of TGFbeta signaling does interfere with GDNF-, but not NRTN-mediated signaling and survival. We propose a model taking into account data from GFRalpha1 crystallization and ontogenetic development of the CG that may explain the differences in TGFbeta-dependence of GDNF and NRTN.
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http://dx.doi.org/10.1111/j.1471-4159.2007.04962.xDOI Listing
December 2007

GDNF applied to the MPTP-lesioned nigrostriatal system requires TGF-beta for its neuroprotective action.

Neurobiol Dis 2007 Feb 1;25(2):378-91. Epub 2006 Dec 1.

IZN, Department of Neuroanatomy, University of Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany.

GDNF is a potent neurotrophic factor for nigrostriatal dopaminergic neurons in vitro and in animal models of Parkinson's disease (PD), but has largely failed when tested in therapeutic applications in human PD. We report here that GDNF requires transforming growth factor-beta (TGF-beta) to elicit its neurotrophic activity. Lesioning the mouse nigrostriatal system with MPTP significantly upregulates striatal TGF-beta2 mRNA levels. As expected, GDNF protects against the destructive effects of MPTP, including losses of TH-ir nigral neurons, striatal dopamine and TH-ir fibers. Application of antibodies neutralizing all three TGF-beta isoforms to the MPTP-lesioned striatum abolishes the neurotrophic effect of GDNF. We show that TGF-beta antibodies are not toxic and do not interfere with retrograde transport of iodinated GDNF, suggesting that TGF-beta antibodies do not impair internalization and retrograde trafficking of GDNF. We conclude that striatal TGF-beta may be essential for permitting exogenous GDNF to act as a neuroprotective factor.
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http://dx.doi.org/10.1016/j.nbd.2006.10.005DOI Listing
February 2007

Transforming growth factor beta2 is released from PC12 cells via the regulated pathway of secretion.

Mol Cell Neurosci 2003 Jan;22(1):75-86

Neuroanatomy and Center for Neurosciences (IZN), University of Heidelberg, Im Neuenheimer Feld 307, 2.OG, D-69120 Heidelberg, Germany.

Transforming growth factor beta2 (TGF-beta2), a prototypic member of a large superfamily of multifunctional cytokines, is expressed by neurons and glial cells. Its subcellular compartmentalization and release from neurons, however, are largely unknown. Here we show that TGF-beta2 colocalizes with the trans-Golgi network marker TGN38 and a marker molecule for secretory granules, chromogranin B (CgB), in PC12 cells. Similarly, primary hippocampal neurons show colocalization of TGN38 and TGF-beta2. A substantial amount of endogenous as well as transfected TGF-beta2 in PC12 cells comigrates with CgB on an equilibrium gradient, suggesting costorage in secretory granules. TGF-beta biological activity peaks in identical fractions. Depolarization of PC12 cells with high potassium triggers colocalization of CgB and TGF-beta2 at the cell surface, suggesting their regulated corelease from secretory granules. High potassium also liberates biologically active TGF-beta from PC12 cells and primary neurons. Our results indicate that a substantial portion of TGF-beta2 is secreted by the regulated secretory pathway in PC12 cells and hippocampal neurons.
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http://dx.doi.org/10.1016/s1044-7431(02)00023-4DOI Listing
January 2003