Published OnlineFirst January 22, 2019; DOI: 10.1158/1078-0432.CCR-18-2380 Translational Cancer Mechanisms and Therapy Clinical Cancer Research L-Carnitine–Mediated Tumor Cell Protection and Poor Patient Survival Associated with OCTN2 Overexpression in Glioblastoma Multiforme Matthias A. Fink1,2, Heiko Paland1,2, Susann Herzog1, Markus Grube1, Silke Vogelgesang3, Kerstin Weitmann4, Angela Bialke5, Wolfgang Hoffmann4, Bernhard H. Rauch1, Henry W.S. Schroeder2, and Sandra Bien-Moller€ 1,2 Abstract Purpose: Apoptotic dysregulation, redox adaptive investigate inhibition of the OCTN2/LC system on in vivo mechanisms, and resilience to hypoxia are major causes of GBM growth. glioblastoma (GBM) resistance to therapy. Commonly Results: Compared with healthy brain, OCTN2 expression known as crucial factors in energy metabolism, OCTN2 was increased in primary and even more so in recurrent GBM (SLC22A5) and its substrate L-carnitine (LC) are increas- on mRNA and protein level. High OCTN2 expression was ingly recognized as actors in cytoprotection. This study associated with a poor overall patient survival; the unadjusted provides a comprehensive expression and survival analysis HR for death was 2.7 (95% CI, 1.47–4.91; P < 0.001). LC of the OCTN2/LC system in GBM and clarifies the system's administration to GBM cells increased their tolerance toward impact on GBM progression. cytotoxicity, whereas siRNA-mediated OCTN2 silencing led to Experimental Design: OCTN2 expression and LC content a loss of tumor cell viability. In line herewith, OCTN2/LC were measured in 121 resected human GBM specimens and inhibition by meldonium resulted in reduced tumor growth 10 healthy brain samples and analyzed for prognostic in an orthotopic GBM mouse model. significance. Depending on LC administration, the effects Conclusions: Our data indicate a potential role of the of hypoxic, metabolic, and cytotoxic stress on survival and OCTN2/LC system in GBM progression and resistance to migration of LN18 GBM cells were further studied in vitro. therapy, and suggests OCTN2 as a prognostic marker in Finally, an orthotopic mouse model was employed to patients with primary GBM. Introduction mechanisms underlying the resistance of GBM cells remain largely elusive, leading to a lack of substantial progress in the therapeutic fi Glioblastoma (GBM), classi ed as WHO grade IV glioma, management of GBM. Several potential reasons exist as to how represents the most frequent and most aggressive type of primary these tumors acquire treatment resistance, including enhanced brain tumor in adults. Despite multimodal therapy including expression of drug efflux transporters, alterations in drug metab- surgical resection followed by adjuvant radiation and chemother- olism, mutations of drug targets, and the activation of survival or apy, GBMs are characterized by rapid tumor recurrence resulting inactivation of death signaling pathways (2, 3). Furthermore, – in a poor prognosis with a median survival of only 12 15 studies argue for a potential role of altered redox homeostasis months (1). Although a number of genetic and epigenetic altera- and energy metabolism in the development of antitumoral drug tions were discovered in recent decades, many of the molecular resistance (4, 5). In recent years, evidence has emerged suggesting that the amino acid derivate L-carnitine (trimethylamine-b-hydroxybutyrate, þ 1Department of Pharmacology/C_DAT, University Medicine Greifswald, Greifs- LC), which is transported into the cells mainly by the Na - wald, Germany. 2Department of Neurosurgery, University Medicine Greifswald, dependent transporter OCTN2 (SLC22A5), contributes to cell 3 Greifswald, Germany. Institute of Pathology, Department of Neuropathology, protection by interacting with various targets inside the cell. 4 University Medicine Greifswald, Greifswald, Germany. Institute for Community Besides its key role in energy metabolism, several studies attest Medicine, University Medicine Greifswald, Greifswald, Germany. 5Independent Trusted Third Party, University Medicine Greifswald, Greifswald, Germany. the OCTN2/LC system a broad spectrum of capabilities including scavenging free radicals, stabilizing membranes, enhancing Note: Supplementary data for this article are available at Clinical Cancer antioxidative resources, and promoting antiapoptotic path- Research Online (http://clincancerres.aacrjournals.org/). ways (6–9). Therefore, the OCTN2/LC system has been widely M.A. Fink and H. Paland contributed equally to this article. discussed as a possible treatment adjunct in some neurodegen- Corresponding Author: Sandra Bien-Moller,€ Department of Pharmacology/ erative disorders such as Parkinson's and Alzheimer's disease to C_DAT, University Medicine Greifswald, Felix-Hausdorff-Str. 3, Greifswald stabilize cellular integrity of compromised neuronal cells (10, 11). 17487, Germany. Phone: 4938-3486-5646; Fax: 4938-3486-5631; E-mail: However, it has not yet been investigated whether GBM cells may [email protected] also benefit from the OCTN2/LC system to increase their surviv- doi: 10.1158/1078-0432.CCR-18-2380 ability, and whether OCTN2 and LC could be prognostic markers Ó2019 American Association for Cancer Research in GBM progression. www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst January 22, 2019; DOI: 10.1158/1078-0432.CCR-18-2380 Fink et al. aimed to investigate the influence of LC on GBM cell survival and Translational Relevance migration in vitro to verify its underlying cytoprotective capabil- Glioblastoma (GBM) is highly resistant to treatment, largely ities. To finally assess the OCTN2/LC system as a therapeutic due to disease heterogeneity and resistance mechanisms. target, we analyzed the influence of the OCTN2/LC inhibitor Understanding the mechanisms that generate resistance is meldonium on in vivo GBM growth using an orthotopic mouse essential for developing more effective treatment strategies. model. Recent studies provide evidence that OCTN2 and its substrate L-carnitine (LC) function as a cytoprotective system that strengthens cellular robustness. Here, we report on the expres- Materials and Methods sion and prognostic impact of the OCTN2/LC system in Human samples resected specimens of patients with newly diagnosed and Following an institutional review board–approved protocol (in recurrent GBM compared with healthy brain. We identified accordance with the ethical standards of the Helsinki Declaration a high OCTN2 expression profile to correlate with a poor of the World Medical Association), fresh human GBM tissues were prognosis in patients with primary GBM, especially in those obtained from patients undergoing surgical resection or biopsy with a holistic therapeutical approach (total tumor resection, within their therapeutic regimen at the Department of Neurosur- radiochemotherapy according to the Stupp protocol). The gery, University Medicine Greifswald (Greifswald, Germany), in mechanistic studies indicate that inhibition of the OCTN2/ the period from April 2007 to August 2016. At our university LC system could reduce survival of GBM cells through hospital, approximately 2 patients with glioblastoma are under- enhanced sensitivity to exogenous influences such as hypoxic, going surgery every month. Because written informed consent metabolic, and cytotoxic stress. Furthermore, the OCTN2/LC was mandatory for both cryopreservation of the specimens and inhibitor meldonium diminished in vivo tumor growth in an obtainment of vital status from official population registry, a total orthotopic GBM mouse model. In summary, our study stresses of 121 patients finally served as cohort for this study. Baseline the role of OCTN2/LC as an actor in GBM cytoprotection, epidemiologic and clinical characteristics of all patients are shown representing a potential target for clinical therapies aimed to in Table 1. On the basis of histologic confirmation according to slow the growth and progression of GBM. the 2007 WHO Classification of Tumors of the CNS (24), resected specimens included 80 primary GBM and 41 recurrences of primary GBM. Furthermore, 24 tumor samples from patients with astrocytoma grade II or III were included in this study. Eight LC-mediated cytoprotection is closely linked to elevated levels nonmalignant brain tissues (frontal and temporal lobes) were of glutathione peroxidase, catalase and superoxide peroxi- obtained by routine autopsies. The patients died of pneumonia, dase (12–14), and to the induction of the NRF2 (nuclear factor heart failure, sepsis or pancreatic cancer, but had no underlying erythroid 2-like) transcriptional network, one of the major path- brain disease. In addition, 2 further nonmalignant brain samples ways used by both normal and cancerous cells to counteract (1 frontal and 1 temporal) were obtained from the BioChain oxidative insults (15). Given that GBM has a high proliferation Institute Inc.. All resected tumor samples and control brain tissues rate combined with an elevated basal metabolic turnover and were cut and frozen at À80 C. formation of reactive oxygen species (ROS) as a natural byprod- uct (16), cancer cells may upregulate their antioxidant resources Cell lines and survival pathways to prevent irreversible cellular dam- The human GBM cell lines A-172, GaMG, HF66, U251MG, age (17, 18). Depending on its pleiotropic effects, accumulation U373, LN18, U87MG (bought from the ATCC), the murine cell of LC may strengthen tumor cell resistance