In Germany every year approximately 3000 people are newly diagnosed with a glioblastoma (GBM), the most malignant brain tumor in adults. Despite aggressive multimodal treatment including surgical resection and radiochemotherapy, the median survival time is only 12 to 15 months after diagnosis. The high malignancy of GBM is ascribed to its resistance towards chemotherapy and its invasive growth into the surrounding brain tissue, which explain the ineffectiveness of surgery and radiation. GBM-recurrence often arises from the spreading of tumor cells far away from their original occurrence. To date, there is no defined treatment standard in case of disease progression during or after standard therapy. Recurrent GBM limits life expectancy to only a few weeks. The research group “Molecular Neurooncology” concentrates on projects regarding GBM biology as well as to develop new therapeutic strategies to treat this tumor.
The research projects are or have been funded by the German Cancer Foundation/Mildred Scheel Foundation, German Research Foundation (DFG),Foundation Ulrike Sauer (ISUS), Software AG Foundation, Interdisciplinary Center for Clinical Research Tübingen and the Else-Übelmesser-Foundation .
GLIOMA CELL PROGRESSION IS ASSOCIATED TO MOTILITY
The tumor suppressor gene p53, the “guardian of the genome”, is mutated in nearly 50 % of all tumors. p53 loss of function mutations are an important step in tumorigenesis and play a pivotal role in the development of cell death resistance, therefore also in the development of resistance towards chemotherapeutic drugs or irradiation. The restauration of p53 activity in glioma cells was suggested to be a promising approach for cancer gene therapy. The chimeric tumor suppressor 1 (CTS-1) is an artificial protein based on the structure and sequence of p53, but in contrast to p53, CTS-1 will not be inactivated or destroyed via proteasomal degradation, in this regard CTS-1 could also be named “dominant-positive p53”. CTS-1 induces cell death both in p53 mutant and p53 wildtype glioma cells. To identify new genes involved in the development of cell death resistance, we generated CTS-1resistant glioma cells and performed a whole genome expression analysis in CTS-1sensitive versus CTS-1resistant sibling cells. Approximately 1000 genes were differentially expressed. Many of these genes are involved in tumor associated processes such as migration, proliferation or apoptosis. In our analyses we found that the expression of many of these differentially expressed genes is regulated by NFκB. In addition, the CTS-1 dependent cell death was dependent on NFκB activity. In this context, we also analyzed the function of IκBζ, an untypical member of the inhibitor of NFκB” (IκB) family, a protein that was highly upregulated in CTS-1 resistant cells and that is suggested to regulate NFκB activity. We demonstrated that in glioma cells, IκBζ expression is induced by irradiation and upregulates the secretion of inflammatory cytokines by these cells. Overexpression of IκBζ in glioma specimen is correlated worse prognosis of glioma patients. Together with our collaboration partner Prof. Mittelbronn in Luxembourg we identified “Carboxypepidase E” (CPE) as a gene that is differentially expressed in CTS-1 resistant versus sensitive glioma cells. A secreted version of CPE provides anti-migratory, but pro-proliferative effects in glioma cells and is suggested to be a so called “switch factor” involved in the decision of a glioma cell either to stay and to proliferate or to migrate and to invade the healthy brain, if the living conditions in the tumor micro-milieu are suboptimal. CPE modulate the expression of EMT proteins in glioma cells, this way mitigating cell migration. Additionally, CPE regulates the metabolic switch toward glycolysis the tumor cell induces to escape starvation. Since glycolysis is associated to tumor cell motility, by this mechanism CPE also inhibits cell motility. Another study we finalized end of 2020 targeted to identify epigenetic modifications that regulate the expression of the MTUS1 (Microtubule Associated Tumor Suppressor) gene. Additionally, we enlightened the role of MTUS1 in the regulation of glioma cell motility, proliferation and induction of cell death. In glioma MTUS1/ATIP1 serves as a tumor suppressor gene and can be used as a prognostic biological marker that is correlated with glioma malignancy and recurrence. Additionally, MTUS1/ATIP1 expression predicts therapy outcome and survival. In glioma cell lines, glioma sphere cultures (GSC), high-grade glioma (HGG) and especially in glioma recurrence, ATIP1 expression is downregulated, probably by promoter hypermethylation. In its function as a tumor suppressor, ATIP1 provides multiple tumor-suppressive functions like mitigating proliferation, cell motility and clonogenic survival. However, ATIP1 is an important player in DNA repair processes and this might interfere with DNA-damaging tumor therapies. In glioma cells, elevated ATIP1 levels push DNA repair and protect the cells from irradiation-induced DNA damage. Therefore, it should be kept in mind that in HGG patients that possess highly ATIP1 positive tumors, even being ATIP1-correlated with a better basal outcome, the elevated expression of ATIP1 might interfere with the anti-tumoral effects of irradiation.
Figure Legend:A simplified model of the role of ATIP1/MTUS1 in tumor progression. B. By activiating double strand DNA repair, MTUS1/ATIP1 impacts the effect of tumor irradiation in glioma.
Höring, E., Harter, P. N., Seznec, J., Schittenhelm, J., Bühring, H.J., Bhattacharyya, S., von Hattingen, E., Zachskorn, C., Mittelbronn, M., Naumann, U. The "go or grow" potential of gliomas is linked to the neuropeptide processing enzyme carboxypeptidase E and mediated by metabolic stress. Acta Neuropathol. (2012) 124:83-97
Brennenstuhl. H., Armento, A., Braczynski, A.K., Mittelbronn, M., Naumann, U. In glioma cells, IκBζ, an atypical member of the inhibitor of nuclear factor kappa B (NFkB) family, is induced by gamma irradiation, regulates cytokine expression and is associated with bad prognosis. Int. J. Oncol. (2015) 47:1971-1980
Ilina, E.I., Armento, A., Garea Sanchez, L., Reichlmeir, M., Braun, Y., Penski C., Capper, D., Sahm, F., Jennewein, L., Harter, P.N., Zukunft, S., Fleming, I., Schulte, D., Le Guerroué, F., Behrends, C., Ronellenfitsch, M.W., Naumann, U., Mittelbronn, M. Effects of soluble CPE on glioma cell migration are associated with mTOR activation and enhanced glucose flux, Oncotarget. 2017 Jun 27. doi: 10.18632/oncotarget.18747
Armento, A., Ilina, E.I., Kaoma, T., Muller, A., Vallar, L., Niclou, S.P., Krüger, MA., Mittelbronn, M. Naumann, U. Carboxypeptidase E transmits its anti-migratory function in glioma cells via transcriptional regulation of cell architecture and motility regulating factors. Int. J. Oncol. 2017, 51: 702-714, (DOI: 10.3892/ijo.2017.4051)
Ranjan N, Pandey V, Panigrahi MK, Klumpp L, Naumann U *, Phanithi PB* The tumor suppressor MTUS1/ATIP1 modulates tumor promotion in glioma: association with epigenetics and DNA repair. Special Edition Recurrent Glioblastoma. Cancers 2021, 13(6), 1245; https://doi.org/10.3390/ cancers13061245 (*) equal authorship
Granting:German Cancer Foundation, Interdisciplinary Center for Clinical Research Tübingen; German Academic Exchange program (DAAD),
(Prof. Dr. U. Naumann)
THERAPEUTIC AND FUNCTIONAL EFFECTS OF VISCUMINS IN GLIOBLASTOMA
In several countries mistletoe lectin (ML) containing drugs have been used in complementary cancer therapy for a long time. ML 1-3 and viscotoxins present in extracts from the mistletoe are the main active components translating anti-cancer activity. Viscotoxins are small peptides inducing cytotoxicity whereas MLs are glycosylated proteins of two subunits of which the α-chain serves as a type I ribosomal inhibitor (RIP). Viscumine/ML-1 is meanwhile known to be the major anticancer compound. We and others have shown that in cancer cells mistletoe based drugs not only block protein translation as predicted by the ML´s function as RIPs, but also provide multimodal anticancer functions. In clinical trials the treatment of cancer patients with ML containing dugs lead to an improvement in the quality of life and a tendency for prolonged survival. Even after intense research until today it is not completely understood how this drugs transmit their anti-tumoral functions. In our study we evaluated the anticancer effects of ISCADOR Qu, an extract of the mistletoe growing on oak trees containing high amounts of MLs as well as those of Aviscumine, a recombinant, but non-glycosylated ML-1 and of purified, naturally glycosylated Viscumine/ML-1. In glioma cells MLs provide several anti-tumoral properties as treatment of these cells with MLs mitigated glioma cell migration, blocked cell division, induced cell death, reduced their immunosuppressive activity and boosted the T- and NK-cell mediated cell killing of glioma cells. Additionally, MLs work in synergy with glioma based standard therapies like irradiation and temozolomide based chemotherapy. In vivo concomintant ML treatment prolonged the survival of glioma bearing mice in both immunocompetent mice harboring syngeneic orthotopically growing gliomas as well as in immunocompromised nude mice bearing othotopically growing human glioma. Taken together we suggest that it might be beneficial to use mistletoe-based drugs as adjuvant therapeutics in GBM patients.
Schötterl S, Miemietz JT, Ilina EI, Wirsik NM, Ehrlich I, Gall A, Huber SM, Lentzen H, Mittelbronn M., Naumann U. “An Assessment of Mistletoe-based drugs work in synergy with radio-chemotherapy in the treatment of glioma in vitro and in vivo in glioblastoma bearing mice. In Technological Innovation in Pharmaceutical Research, Book Publisher International 2021, ISBN: 978-93-90206-65-0, E-ISBN: 978-93-90206-66-7 (in press)
Schötterl S, Naumann U. Antitumoral effects of mistletoe-based preparations in the treatment of experimental glioma Die Mistel in der Tumortherapie V, KVC-Verlag Essen ISBN: 9783965620308, 2020, 79-84
Schötterl S, Miemietz JT, Ilina EI, Wirsik NM, Ehrlich I, Gall A, Huber SM, Lentzen H, Mittelbronn M, Naumann U. Mistletoe-Based Drugs Work in Synergy with Radio-Chemotherapy in the Treatment of Glioma In Vitro and In Vivo in Glioblastoma Bearing Mice. Evid Based Complement Alternat Med. Volume 2019, Article ID 1376140, 17 pages,https://doi.org/10.1155/2019/1376140
Schötterl S, Hübner M, Armento A, Veninga V, Wirsik NM, Bernatz S, Lentzen H, Mittelbronn M., Naumann U. Mistletoe lectin I reduces glioma cell motility by changing mainly the expression of genes assoiated to TGF-β signaling. Phytomedicine2019:61, Suppl 1, doi: 10.1016/j.phymed.2019.09.122
Schötterl S, Huber SM, Lentzen H, Mittelbronn M, Naumann U. Adjuvant therapy using mistletoe containing drugs boosts the T-cell-mediated killing of glioma cells and prolongs the survival of glioma-bearing mice. Evid Based Complement Alternat Med. 2018 vol. 2018, Article ID 3928572, 12 pages, 2018. https://doi.org/10.1155/ 2018/3928572/
Schötterl S, Hübner M, Armento A, Veninga V, Wirsik NM, Bernatz S, Lentzen H, Mittelbronn M, Naumann U Viscumins functionally modulate cell motility associated gene Expression. Int. J. Oncol. 2017, 50: 684-696
ONCOLYTIC VIRUSES TO TREAT GBM
Preclinical and clinical trials have demonstrated that viruses can be used as potent agents in the treatment of cancer, also for the treatment of glioma. These so called oncolytic viruses (OV) of different origin can replicate in and subsequently kill tumor cells, but not non-neoplastic cells. Additionally, OVs can contain therapeutic genes triggering either the patient´s anti-tumor immune response, modulating the GBM microenvironment, or coding for prodrug suicide genes. However, the clinical efficacy of GBM oncovirotherapy has not yet achieved the promising preclinical laboratory results. To address this mismatch, one should keep in mind the complex interaction between cancer cells, OV infection and replication, the adjacent tumor microenvironment, chemotherapy as well the patient´s immune system, indicating that not only OVs play a role in an efficient (onco)lysis of GBM cells. There is also strong evidence that OVs are crucial inducers of anti-tumor immune responses and might tilt the suppressive effects of immune evasion mechanisms induced by GBM cells by several mechanisms. Overall, OVs might drive anti-GBM immune responses and can initiate anti-GBM immunity.
In a close and long lasting collaborative project with the virologist Prof. PS Holm (TU Munich) we analyzed the therapeutic impact of a YB-1 dependent oncolytic adenovirus (YB-1-OAV) in vitro as well as in glioma bearing mice. We have shown that YB-1 is expressed in glioma cells, glioma stem cells (GSC) as well as in GBM tissue, especially in recurrent glioma, whilst it is absent in non-neoplastic cells or tissue of the brain as well as in immune cells. YB-1-OAV replication is dependent on nuclear YB-1, and in consequence, YB-1-OAV infects and lyses glioma cells as well as therapy resistant GSCs, but leaves primary and immortalized non-tumor cells like astrocytes or human immune cells unattached. Injection of orthotopically growing GBM derived from chemotherapy resistant GSCs with YB-1-OAV prolonged the median survival of mice significantly, whereas the chemotherapeutic drug Temozolomide (TMZ) had no impact on survival. Brain and lung injections of YB-1-OAV in syrian hamsters, a species susceptible for adenoviral infection and replication, demonstrated that YB-1-OAV is safe since no signs of toxicity as well as no virus replication and virus offspring was detectable in any tissue.
To further optimize oncovirotherapy of GBM we examined the effects of glioma irradiation on YB-1-OAV based therapy. Irradiation induces an upregulation of YB-1 expression as well as its nuclear translocation, in consequence enhances virus replication in and lysis of GBM cells. Using a human brain slice culture modell, we demonstrated that irradiation prior to OAV treatment leads to enhanced tumor cell lysis, reduced the growth and tumor cell infiltration into the healthy tissue. Additionally, this combination therapy prolonged the survival of glioma bearing mice.
Since OAVs have to be delivered intratumorally and therefore do not necessarily hit those tumor cells that have migrated away from the original tumor and are located far away from the virus injection side, we recently started, in collaboration with PD Dr. Lusine Danielyan (Pharmacology, Tübingen), a project that aims to develop an approach that uses intranasal delivery of shuttle cells loaded with YB-1-OAV as vehicles for the virus transport towards infiltrating glioma cells. Meanwhile we showed that the shuttle cells were efficially attracted by the tumor and reached the location of the tumor and infiltrating tumor cells about 72 h after intranasal application. In further experiments the shuttle cell will be loaded with OAV and the theraputical impact of the INA-based oncolytic virotherapy will be evaluted.
Figure Legend: Shuttle cells (red) are attracted by glioma cells (green) when appllied intranasally. Pictures were taken 72 h after cell administration.
In a further approach to optimize oncolytic virotherapy of GBM, we armed YB-1-OAV to express an immune checkpoint inhibitor and will determine the immune-stimulatory effect by combining oncovirotherapy with immunotherapy.
Czolk R, Schwarz N, Koch H, Schötterl S, Wuttke TV, Holm PS, Huber SM, Naumann U. In brain tumor initiating cells, irradiation enhances the therapeutic effect of the oncolytic adenovirus XVir-N-31. Int J Mol Med 2019 (in press)
Mantwill, K, Naumann, U., Seznec, J., Girbinger, V., Lage, H. Surowiak P., Beier, C., Mittelbronn, M., Schlegel, J, Holm, P.S. 2013. YB-1 dependent oncolytic adenovirus efficiently inhibits tumor growth of glioma cancer stem like cells. Translational Med. 2013, 11:216
Naumann, U., Holm, P.S. Oncovirotherapy of glioblastoma – a kind of immunotherapy? Brain Disorders and Therapy( 2015), dx.doi.org/10.4172/2168-975X.S2-001
Granting: BMBF (Holm), Else-Übermesser-Stiftung (Naumann), German Research Foundation (Holm, Naumann). German Cancer Foundation (Naumann, Danielyan)
THE FUNCTION OF PERICYTES IN GBM NEOANGIOGENESIS
A key hallmark of GBM is the generation of new blood vessels that are on the one hand a central diagnostic feature and on the other hand a current target for treatment strategies. One pathological hallmark that distinguishes GBM from lower grade glioma is its abundant and aberrant vasculature. The malformed GBM vasculature is accompanied by vessel permeability and the breakdown of the blood-brain barrier (BBB).
We recently identified that pericytes are the major cell type of newly formed blood vessels within glioblastoma, the so-called vascular proliferations. However, the formation of those vascular proliferations is poorly understood. Our research of the last years has been shown that the upregulation of central epithelial-to-mesenchymal transcription factors (EMT) such as Slug and Twist strongly reflects the amount of neo-angiogenesis in both pilocytic astrocytomas WHO grade I and GBM WHO grade IV. Furthermore, we identified non-neoplastic pericytes as unique source of expression of EMT factors in astrocytic brain tumors. This sheds a new light on the EMT program in human gliomas indicating that not neoplastic glial cells but pericytes are the source of EMT transcription factors. In this GBM-vessel-associated pericytes (GA-Peris) the observed elevated SLUG expression, paralleled by the induction of cell proliferation and cell migration, by the induction of metabolic changes and by an altered growth morphology, is induced by TGF-β, a cytokine highly expressed by GBM cells. SLUG expression in pericytes is important for both proliferation as well as migration of pericytes at least in cell culture, since neutralization of TGF-β or knocking down SLUG mitigated or even abolished proliferation, cell motility, metabolic activity and morphological alterations in primary human brain microvascular pericytes (HBVP). Our data indicate that GBM cells actively modulate neovascularization not only by modulating endothelial cells, but also by influencing the function and characteristics of adjacent vessel-associated pericytes. This process might be responsible for the formation of an unstructured tumour vasculature. To better understand how glioma secreted TGF-β and elevated pericytic SLUG-expression modulate the function of pericytes during neoangiogenic processes in GBM we focused our interest on how the above mentioned factors influence the vascular structure in the tumor area and how they are involved in the GBM associated leakiness of the BBB.
Mäder L, Blank AE, Capper D, Jansong J, Baumgarten P, Wirsik NM, Zachskorn C, Ehlers J, Seifert M, Klink B, Liebner S, Niclou S, Naumann U, Harter PN, Mittelbronn M. Pericytes/vessel-associated mural cells (VAMCs) are the major source of key epithelial-mesenchymal transition (EMT) factors SLUG and TWIST in human glioma. Oncotarget. 2018 May 8;9(35):24041-24053.
Wirsik NM, Ehlers J, Mäder L, Ilina EI, Blank AE, Grote A, Feuerhake F, Baumgarten P, Devra j K, Harter PN, Mittelbronn M*, Naumann U*. TGF-β activates pericytes via induction of the epithelial to mesenchymal transition protein SLUG in glioblastoma. Neuropathol Appl Neurobiol. 2021 Mar 29. doi: 10.1111/nan.12714. Online ahead of print.PMID: 33780024 (*) equal authorship
Funding: Interdisciplinary Center for Clinical Research Tübingen, Chinese Scholarship Council
- Schumacher L, Slimani R, Zizmare L, Ehlers J, Kleine Borgmann F, Fitzgerald JC, Fallier-Becker P, Beckmann A, Grißmer A, Meier C, El-Ayoubi A, Devraj K, Mittelbronn M, Trautwein C, Naumann U. TGF-Beta Modulates the Integrity of the Blood Brain Barrier In Vitro, and Is Associated with Metabolic Alterations in Pericytes. Biomedicines 2023, 11, 214. doi.org 10.3390/biomedicines11010214
- Klawitter M, El-Ayoubi A, Buch J, Rüttinger J, Ehrenfeld M, Lichtenegger E,. Krüger MA, Mantwill K, Koll FJ, Kowarik MC, Holm PS, Naumann U. The oncolytic adenovirus XVir-N-31, in combination with the blockade of the PD-1/PD-L1 axis, conveys abscopal effects in a humanized glioblastoma mouse model. Int. J. Mol. Sci.2022, Volume 23, Issue 17, 9965
- Wirsik NM, Ehlers J, Mäder L, Ilina EI, Blank AE, Grote A, Feuerhake F, Baumgarten P, Devra j K, Harter PN, Mittelbronn M*, Naumann U*. TGF-β activates pericytes via induction of the epithelial to mesenchymal transition protein SLUG in glioblastoma. Neuropathol Appl Neurobiol. 2021 Mar 29. doi: 10.1111/nan.12714. (*) equal authorship
- Ranjan N, Pandey V, Panigrahi MK, Klumpp L, Naumann U*, Phanithi PB* The tumor suppressor MTUS1/ATIP1 modulates tumor promotion in glioma: association with epigenetics and DNA repair. Special Edition Recurrent Glioblastoma. Cancers 2021, 13(6), 1245 (*) equal authorship
- Reautschnig P, Wahn N, Wettengel J, Schulz A, Latifi N, Vogel P, Kang TW, Zarges C, Naumann U, Zender L, Li JB, Stafforst T. CLUSTER guide RNAs enable precise and efficient RNA editing in cell culture and in vivo, Nature Biotechnology 2022. doi: 10.1038/s41587-021-01105-0.
- Kemmerer CL, Schittenhelm J, Dubois E, Neumann L, Häsler LM, Lambert M, Renovanz M, Kaeser SA, Tabatabai G, Ziemann U, Naumann U, Kowarik MC. Cerebrospinal fluid cytokine levels are associated with macrophage infiltration into tumor tissues of glioma patients. BMC Cancer 2021;21(1):1108. doi: 10.1186/s12885-021-08825-1
- Czolk R, Schwarz N, Koch H, Schötterl S, Wuttke TV, Holm PS, Huber SM, Naumann U. In brain tumor initiating cells, irradiation enhances the therapeutic effect of the oncolytic adenovirus XVir-N-31. Int J Mol Med; 2019 Oct;44(4):1484-1494
- Schötterl, S., Hübner, M., Armento, A., Veninga, V., Wirsik, N.M., Bernatz, S., Lentzen, H., Mittelbronn, M., Naumann, U. Viscumins functionally modulate cell motility associated gene Expression. Int. J. Oncol. 2017, 50: 684-696
- Ilina, E.I., Armento, A., Garea Sanchez, L., Reichlmeir, M., Braun, Y., Penski C., Capper, D., Sahm, F., Jennewein, L., Harter, P.N., Zukunft, S., Fleming, I., Schulte, D., Le Guerroué, F., Behrends, C., Ronellenfitsch, M.W., Naumann, U., Mittelbronn, M. Effects of soluble CPE on glioma cell migration are associated with mTOR activation and enhanced glucose flux, Oncotarget. 2017 Jun 27. doi: 10.18632/oncotarget.18747
- Armento, A., Ilina, E. I., Kaoma, T., Muller, A., Vallar, L., Niclou, S.P., Krüger, MA., Mittelbronn, M. Naumann, U. Carboxypeptidase E transmits its anti-migratory function in glioma cells via transcriptional regulation of cell architecture and motility regulating factors. Int. J. Oncol. 2017, 51:702-714
Sascha Venturelli, Physiology, UKT Tübingen
Lusine Danielyan, Pharmacology, University of Tübingen
Michel Mittelbronn, Neuropathologie, Luxembourg Centre of Neuropathology (LCNP), Luxembourg
Per Sonne Holm, Experimentelle Tumortherapie, Urologie, Klinikum Rechts der Isar, TU München; Molekulare Onkologie, Universität Insbruck, Österreich
Bernd Pichler, Institut für Präklinische Bildgebung und Radiopharmazie, Universität Tübingen
Robert Feil, Signaltransduktion, Transgene Modell, IFIB, Tübingen
Karin Schilbach, Hämatologie, Kinderklinik, UKT Tübingen
Stephan Huber, Radioonkologie, UKT Tübingen
Thorsten Stafforst, Interfakultäres Institut für Biochemie, Tübingen
XVir Therapeutics GmbH, München
NOA: Neuroonkologische Arbeitsgemeinschaft (NOA ) in der Deutschen Krebsgesellschaft
ZNO: Zentrum für Neuroonkologie (ZNO)
Deutsche Krebshilfe: http://www.krebshilfe.de
DFG; DFG - Deutsche Forschungsgemeinschaft
Software AG Stiftung: www.sagst.de/
Innovationsstiftung Sauer: www.isus-stiftung.de/