Theranostics 2021; 11(16):7844-7868. doi:10.7150/thno.58655 This issue

Research Paper

GLS-driven glutamine catabolism contributes to prostate cancer radiosensitivity by regulating the redox state, stemness and ATG5-mediated autophagy

Anna Mukha1,2*, Uğur Kahya1,2*, Annett Linge1,3,4,5,6, Oleg Chen1,7, Steffen Löck1,3,4,5, Vasyl Lukiyanchuk1,2, Susan Richter8, Tiago C. Alves9, Mirko Peitzsch8, Vladyslav Telychko1, Sergej Skvortsov10,11, Giulia Negro10,11, Bertram Aschenbrenner10,11, Ira-Ida Skvortsova10,11, Peter Mirtschink8, Fabian Lohaus1,3,4,5, Tobias Hölscher1,5,6, Hans Neubauer12, Mahdi Rivandi12, Vera Labitzky13, Tobias Lange13, André Franken12, Bianca Behrens14, Nikolas H. Stoecklein14, Marieta Toma15,16, Ulrich Sommer16, Sebastian Zschaeck1,3,4,5, Maximilian Rehm1,3,4,5, Graeme Eisenhofer8, Christian Schwager4,17,18,19,20, Amir Abdollahi4,17,18,19,20, Christer Groeben21, Leoni A. Kunz-Schughart1,6, Gustavo B. Baretton16, Michael Baumann1,4,5, Mechthild Krause1,2,3,4,5,6, Claudia Peitzsch1,3,4,6#✉, Anna Dubrovska1,2,3,4,6#✉

1. OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden and Helmholtz-Zentrum Dresden-Rossendorf, Germany
2. Institute of Radiooncology - OncoRay, Helmholtz-Zentrum Dresden-Rossendorf (HZDR) Dresden, Germany
3. German Cancer Consortium (DKTK), Partner Site Dresden, Germany
4. German Cancer Research Center (DKFZ), Heidelberg, Germany
5. Department of Radiotherapy and Radiation Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany
6. National Center for Tumor Diseases (NCT), Partner Site Dresden, Germany
7. Department of Cell Signaling, Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine
8. Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany
9. Department for Clinical Pathobiochemistry, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany
10. Department of Therapeutic Radiology and Oncology, Medical University of Innsbruck, Innsbruck, Austria
11. EXTRO-Lab, Tyrolean Cancer Research Institute, Innsbruck, Austria
12. Department of Obstetrics and Gynecology, Medical Faculty and University Hospital of the Heinrich-Heine University Düsseldorf, Germany
13. Institute of Anatomy and Experimental Morphology, Center for Experimental Medicine, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Germany
14. General, Visceral and Paediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
15. Institute of Pathology, University of Bonn, Bonn, Germany
16. Institute of Pathology, Universitätsklinikum Carl Gustav Carus Dresden, Dresden, Germany
17. Heidelberg Ion-Beam Therapy Center (HIT), Department of Radiation Oncology, Heidelberg University Hospital (UKHD), National Center for Tumor Diseases (NCT), Heidelberg, Germany
18. German Cancer Consortium (DKTK) Core Center, Clinical Cooperation Units (CCU) Translational Radiation Oncology and Radiation Oncology, Heidelberg, Germany
19. Heidelberg Institute of Radiation Oncology (HIRO), National Center for Radiation Research in Oncology (NCRO), German Cancer Research Center (DKFZ) and Heidelberg University Hospital (UKHD), Heidelberg, Germany
20. Division of Molecular and Translational Radiation Oncology, Heidelberg Medical Faculty (HDMF), Heidelberg University, Heidelberg, Germany
21. Department of Urology, Medical Faculty Carl Gustav Carus, TU Dresden, Dresden, Germany
* These authors share first authorship
# These authors share senior authorship

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Citation:
Mukha A, Kahya U, Linge A, Chen O, Löck S, Lukiyanchuk V, Richter S, Alves TC, Peitzsch M, Telychko V, Skvortsov S, Negro G, Aschenbrenner B, Skvortsova II, Mirtschink P, Lohaus F, Hölscher T, Neubauer H, Rivandi M, Labitzky V, Lange T, Franken A, Behrens B, Stoecklein NH, Toma M, Sommer U, Zschaeck S, Rehm M, Eisenhofer G, Schwager C, Abdollahi A, Groeben C, Kunz-Schughart LA, Baretton GB, Baumann M, Krause M, Peitzsch C, Dubrovska A. GLS-driven glutamine catabolism contributes to prostate cancer radiosensitivity by regulating the redox state, stemness and ATG5-mediated autophagy. Theranostics 2021; 11(16):7844-7868. doi:10.7150/thno.58655. Available from https://www.thno.org/v11p7844.htm

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Abstract

Graphic abstract

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization.

Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients.

Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy.

Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

Keywords: Prostate cancer, Radioresistance, Cancer stem cells, Autophagy, GLS1