L-Carnitine–Mediated Tumor Cell Protection and Poor Patient Survival Associated with OCTN2 Overexpression in Glioblastoma Multiforme Matthias A

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 2019 American Association for Cancer Research in GBM progression.

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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 80 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 as anticancer treat- line GL261, and patient derived primary GBM cells were used for ments rely, in part, on the destructive effect of ROS in their target the qualitative protein assessment. LN18, a WHO grade IV GBM cells (19, 20). cell line, was further used as in vitro model for the mechanistic The OCTN2/LC system exists in almost all tissues, including studies. Because of its similarity to human GBM growth charac- the brain, as it is involved in essential metabolic processes (21). teristics (25), GL261 cells were selected for our mouse model of Well-known as a key compound in the "carnitine shuttle," LC malignant glioma. Detailed information on all cell lines are facilitates the transport of middle- and long-chain acids into provided in Supplementary Table S1. Only cells at low passages mitochondria to subsequently undergo b-oxidation, allowing were used for the experiments. By employing a PCR-based assay, cellstometabolizefattyacidsasenergyresource(22).Similar all cell lines were routinely monitored for potential Mycoplasma to other tumors, GBMs have long been thought to rely upon contamination. glycolysis for energy production, while current studies suggest that glucose accounts for only <50% of acetyl-CoA production Quantitative real-time PCR analysis in gliomas (23). However, it was shown that fatty acid metab- Total RNA was isolated using PeqGold RNAPure (PeqLab) olism serves as an alternative energy source to promote GBM and reverse transcripted using the High Capacity cDNA progression, whereas inhibition of b-oxidation led to pro- Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative longed survival in a GBM mouse model (5). real-time PCR was performed on a 7900HT Fast Real-Time PCR With a broad spectrum of cytoprotective effects and of meta- system (Thermo Fisher Scientific) using the following Gene bolic relevance, the OCTN2/LC system may be a potential actor in Expression Assays on Demand (Thermo Fisher Scientific): cell resistance, but its role in GBM biology has not yet been OCTN2/SLC22A5, Hs00929869_m1; CPT1A, Hs00912671_m1; explored. Here, we report on the expression and prognostic CPT1C, Hs00380581_m1; and eukaryotic 18S rRNA endogenous impact of the OCTN2/LC system in resected tumor specimens control, 4310893E. Target gene expression was normalized to of patients with newly diagnosed and recurrent GBM. We further 18S rRNA and presented as box plots with the median and the

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Table 1. Clinicopathologic characteristics of the analyzed patient cohort Entire glioma patient cohort Parameters Patient cohort Glioma subtype (n) GBM WHO grade IV 121 Primary GBM 80 Relapses of primary GBM 41 Astrocytoma WHO grade II/III 24

Parameters Patients with primary GBM Relapses of primary GBM Astrocytoma WHO grade II/III Age at diagnosis [years] Median 65 59 37 Min.–Max. 25–83 25–79 1–59 Age classes (n,%) <50 years 7 (8.75) 9 (21.95) 21 (87.5) 50–59 years 24 (30) 11 (26.83) 3 (12.5) 60–70 years 22 (27.5) 15 (36.59) 0 (0) >70 years 27 (33.75) 6 (14.63) 0 (0) Sex (n,%) Male 52 (65) 32 (78) 20 (83.33) Female 28 (35) 9 (22) 4 (16.67)

Patients with primary glioblastoma Parameters Median survival [days/months] Range of survival [days/months] Tumor resection status (n,%) Biopsy 2 (2.5) 50% tumor resection 3 (3.75) Total tumor resection (macroscopic) 42 (52.5) Subtotal tumor resection 26 (32.5) Unknown 7 (8.75) Therapeutic regimen (n,%) RCTx according to Stupp and colleagues 49 (61.25) Radiomonotherapy 26 (32.5) No adjuvant therapy 3 (3.75) Unknown 2 (2.5) Overall survival (n,%) All patients with known vital status 76 (95) 275/9 33–1780/1.1–58.5 Patients with RCTx according to Stupp and colleagues 45 (56.25) 493/16.2 75–1780/2.5–58.5 Patients with radiomonotherapy 26 (32.5) 152.5/4.5 33–992/1.1–32.6 Patients with total tumor resection 37 (46.25) 373/12.3 75–1512/2.5–49.7 Patients with subtotal tumor resection 22 (27.5) 147/4.8 33–617/1.1–20.3 Survival rates (n,%) 1-year survival 35 (43.75) 2-year survival 16 (20) Vital status at study end (08/2016) (n,%) Dead 70 (87.5) Alive 6 (7.5) Unknown 4 (5)

5th/95th percentiles in relation to the average expression in the homogenized tissue suspended in PBS) determined by the control samples using DDCt method. BCA method.

Western blot analysis Generation of hypoxic conditions in cell culture Preparation of primary GBM cells was done as described To achieve hypoxic conditions, LN18 GBM cells were cultivated previously (26). OCTN2-rich placenta membrane vesicles were in a hypoxia chamber (<0.5 Vol.-% O2; GENbox) for 24 to prepared as described elsewhere (27). Preparation of lysates 96 hours using a hypoxia pad (GENbox anaer Generator, Bio- from the investigated cell lines as well as the detailed steps of Merieux) at 37C. frozen tissue sample homogenization, protein measurement, and subsequent immunoblot analysis are described in the Cell viability assays Supplementary Data. Cells were seeded in 96-well multiplates (5,000 cells/well) using 150-mL culture medium containing 0.05% FCS. After Measurement of LC in GBM tissue 24 hours, cells were incubated with LC alone or in combination Free LC was measured in tissue samples using the L-carnitine with etomoxir, temozolomide, or H2O2 undereithernormoxic Colorimetric/Fluorometric Assay Kit (BioVision) according to or hypoxic conditions as described above. Afterwards, medium the manufacturer's protocol. Samples were analyzed on a was replaced by fresh medium containing 10% resazurine TECAN Infinite M200 (Tecan) multimode reader (excitation: (PromoCell) and cells were incubated for 2 hours at 37C, 535 nm, emission: 587 nm). LC concentrations were normal- before cell viability was analyzed on multimode reader ized to the protein content of each sample (measured in the (TECAN Infinite M200, Tecan) by fluorescence measurement

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(excitation: 530 nm; emission: 590 nm). Viability data were icant at , P < 0.05; , P < 0.01; , P < 0.001 level. Data are shown calculated as percentage of solvent-treated cells. as mean SD. The tests used for statistical comparison are indicated in the respective figure legends. Overall survival was OCTN2 silencing using RNAi calculated from the time of diagnosis until death at the last follow- LN18 GBM cells were cultured in 12- and 96-well plates and up. Information on vital status and date of death were obtained transfected with OCTN2 or control siRNA (sc-42560/sc-37007, from official population registry. Santa Cruz Biotechnology, 5 pmol/well) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. To enhance knockdown effectivity siRNA transfection was repeated Results after 24 hours. OCTN2 expression is enhanced in GBM on mRNA and protein level L-[3H]-carnitine accumulation assay Compared with nonmalignant brain (NMB), a stepwise 2.3- to The functional activity of OCTN2 in control and OCTN2- 4.4-fold increase of OCTN2 mRNA expression was measured siRNA–transfected LN18 cells was analyzed by L-[N-methyl- in astrocytoma grade III (AC-III) and GBM specimens, respectively 3H]-carnitine (3H-carnitine, 80Ci/mmol, American Radiolabeled (Fig. 1A), while its expression was not elevated in astrocytoma Chemicals Inc.) uptake assay. Forty-eight hours after transfection grade II (AC-II) samples. Subdivision into primary and relapsed cells were washed using prewarmed PBS and incubated with GBM revealed no significant differences (4.3-fold vs. 4.5- L-[3H]-carnitine in transport buffer (final concentration 1 mCi/ fold, Fig. 1B). In comparison with NMB, OCTN2 mRNA expres- mL, 12,5 nmol/L) for 30 minutes. LC uptake was stopped by sion was similar in U87MG cells, while LN18 cells had 3-fold washing thrice using ice-cold PBS before cells were lysed using higher mRNA levels (Fig. 1B). Immunoblot analysis also revealed 300-mL lysis buffer/well (0.2% SDS, 5 mmol/L EDTA). A total a significant upregulation of OCTN2 on protein level in primary of 150 mL of the lysates were mixed with 2-mL scintillation GBM (34.5-fold) and relapsed tumor samples (37.2-fold; Fig. 1C liquid Rotiszint eco plus (Carl Roth) and analyzed on a and E). LN18 and U87MG had 3.5-fold and about 2-fold higher b-Counter Tri-Carb 2810TR Low Activity Liquid Scintillation OCTN2 protein levels compared with NMB, respectively. Expres- Analyzer (PerkinElmer Inc.). The uptake rates were normal- sion of OCTN2 was detectable in various human glioma cell lines ized to the protein content of each sample lysate. (A-172, GaMG, HF66, LN18, U251MG, U373, U87MG), in a patient-derived culture of primary GBM cells and in the murine Caspase-3 activity assay GL261 cell line. OCTN2 expression was validated by a positive Cells were seeded on 12-well plates (0.1 106 cells/well). After control of high OCTN2–expressing placenta vesicles (Fig. 1D). preincubation with LC or aqua dest, in medium containing 0.05% More detailed information on the used cell lines are provided in FCS for 6 hours, cells were treated with temozolomide, carmus- Supplementary Table S1. tine, vincristine or H2O2 for 48 hours. Afterwards, apoptotic The uptake of LC in cells by OCTN2 is known to depend on activity was studied by measuring caspase-3 activity using the sodium (27). To analyze the extent to which LC-uptake into LN18 Caspase-3 Fluorometric Assay (R&D Systems) according to man- cells is mediated by OCTN2, uptake of tritium-labeled LC was ufacturer's instructions. investigated both in sodium-containing and sodium-free medium as well as in the presence of the OCTN2 inhibitor meldo- Cell migration analysis nium (28). The results demonstrate a sodium-dependent LC Cells were seeded in 24-well plates. After reaching con- uptake via OCTN2, which is sensitive to meldonium (Fig. 1F). fluence, the cell layer was scratched with a 100-mL pipette tip Subsequent correlation analyses revealed a positive association and washed 3 times with PBS. From each scratch 3 randomly between OCTN2 mRNA and protein expression in both primary distributed images were taken with PALM RoboSoftware on GBM samples (Spearman r ¼ 0.5327, P < 0.001; Fig. 1G) and a AxioObserver.Z1 microscope (Zeiss). Afterwards, cells were relapsed tumor specimens (Spearman r ¼ 0.6108, P < 0.001; cultivated with DMEM supplemented with 0.05% FCS, and 5 Fig. 1H). Furthermore, the tissue content of the OCTN2 substrate mmol/L hydroxyurea to prevent cell proliferation. LC was added LC was analyzed. LC concentration was elevated in both pri- for 24 hours. The scratches were analyzed as described above after mary (4-fold) and relapsed GBM (5-fold) compared with NMB 24 hours at the same positions. Finally, the scratch area was (P < 0.001 and P ¼ 0.001, respectively; Fig. 1I), but did not calculated with ImageJ (NIH, Bethesda, MD). correlate with the OCTN2 expression (Fig. 2E and F).

Orthotopic GBM mouse model OCTN2 overexpression correlates with poor patient survival To evaluate OCTN2 as a suitable therapeutic target, we used On the basis of the median OCTN2 mRNA/protein expression, the murine GBM cell line GL261, which was stereotactically the GBM patient cohort was divided into a high and a low implanted into the brain of C57BL/6J mice. The animal expression group. As demonstrated in Fig. 2A and B, a high study has been evaluated and approved according to animal OCTN2 mRNA or protein expression was associated with a welfare guidelines and German laws on animal welfare signiﬁcantly worse survival time. On mRNA level, the median by the Landesveterin€ar- und Lebensmitteluntersuchungsamt survival time for the low OCTN2–expressing group were 528 days Mecklenburg-Vorpommern. The detailed procedures and hous- (17.4 months) compared with 268 days (8.8 months) of the ing conditions are described in the Supplementary Data. high OCTN2–expressing group (Fig. 2A). The unadjusted HR for death was 2.16 (95% CI, 1.25–3.73, P ¼ 0.005) for a high Statistical analysis OCTN2 mRNA expression. For OCTN2 protein expression, Statistical analyses were performed using SPSS 21.0 (IBM) and the survival values were 254 days (8.4 months) for the high GraphPad Prism 6 (GraphPad Software) and considered signif- OCTN2–expressing group and 579 days (19 months) for the low

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ABC 100 100 NMB NMB NMB NMB GBM GBM GBM GBM 128T 128F 115T 115F 086 078 054 061 LN18 * ** *** *** OCTN2 10 * 10 β-Actin NMB GBM GBM GBM GBM GBM GBM GBM GBM 119F 001 003 007 011 009 023 121 109 (arb. unit) 1 (arb. unit) 1 OCTN2 β-Actin

OCTN2 mRNA expression OCTN2 mRNA n n n n = 10 = 10 = 5 = 106 expression OCTN2 mRNA n = 10 n = 70 n = 36 0.1 0.1 NMB GBM GBM GBM GBM GBM GBM GBM GBM NMB AC-II AC-III GBM NMB pr. rel. LN U87 119T 113 117 030 074 049 066 046 051 GBM GBM 18 MG

D OCTN2 pr. GBM U87 U251 Positive U373 LN18 HF66 GaMG A-172 GL261 cells MG MG control Marker xxx x xxxxx Human β-Actin NMB GBM GBM GBM GBM GBM GBM GBM x Murine 129F 054 061 072 073 079 080 068 OCTN2 OCTN2 β-Actin β-Actin EF G *** 1,000 *** 0.8 1,000 Spearman ** ** *** r = 0.5327 P < 0.0001 100 n = 54 100 0.6 10 0.4 10 n.s. 1 (arb. unit) (arb. unit) H-LC uptake 3 1 0.2 0.1 (pmol/mg protein)

n n n Primary GBM

= 10 = 61 = 33 OCTN2 protein expression OCTN2 protein expression 0 0.0 0.01 NMB pr. rel. LN18 U87 Control Sodium Meldonium 0.1 1 10 100 GBM GBM MG free OCTN2 mRNA expression HI

1,000 Spearman 1 r = 0.6108 P = 0.0006 *** n = 28 100 *** 0.1

(arb. unit) 0.01

1 LC (nmol/μL)

Relapsed GBM n = 5 n = 36 n = 24 OCTN2 protein expression 0.1 0.001 0.1 1 10 100 NMB Primary Relapsed GBM GBM OCTN2 mRNA expression

Figure 1. mRNA and protein expression of OCTN2 in glioblastoma in comparison with nonmalignant brain. A, qRT-PCR analyses demonstrating OCTN2 mRNA expression levels in frontal/temporal lobes of nonmalignant brain (NMB, control), astrocytoma grade II (AC-II), astrocytoma grade III (AC-III), and glioblastoma (GBM) specimens. B, OCTN2 mRNA expression in NMB, primary GBM (pr. GBM), relapsed GBM (rel. GBM), and in the GBM cell lines LN18 and U87MG. C, Representative immunoblots of total protein lysates from NMB, GBM, and LN18 cell line analyzed for OCTN2 protein expression, each well representing an individual GBM sample numbered with a 3-digit code (anonymized sample identiﬁcation), NMB: F, frontal lobe; T, temporal lobe. D, Qualitative assessment of OCTN2 protein expression in different GBM cell lines (A-172, GaMG, GL261, HF66, LN18, U251MG, U373, U87MG), patient derived primary (pr.) GBM cells and high OCTN2–expressing placenta vesicles (positive control) determined by immunoblot analysis. Detection of b-actin was used as loading control. E, Densitometric analyses of protein expression determined by Western blot (WB) in GBM in comparison with NMB. F, Uptake of 3H-labeled LC (12.5 nmol/L, 30 minutes) in LN18 cells in sodium- containing and sodium-free buffer (sodium replaced by choline) as well as after treatment with the OCTN2 inhibitor meldonium (100 mmol/L) in sodium- containing buffer, n ¼ 3 (each in triplicates). G and H, Correlation between OCTN2 mRNA and protein expression in primary GBM (G) and relapsed GBM (H; Spearman correlation analysis). I, LC concentration in NMB as well as primary and relapsed GBM specimen normalized to the protein content of each individual sample. Box plots of patient sample data are shown with median and lower/upper quartile, whiskers from 5th to 95th percentile. One-way ANOVA (, P < 0.05; , P < 0.01; , P < 0.001; arb., arbitrary).

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ABKaplan–Meier survival analysis: OCTN2 mRNA Kaplan–Meier survival analysis: OCTN2 protein

1.0 OCTN2 mRNA > median 1.0 OCTN2 protein > median 2-year 1-year 2-year OCTN2 mRNA < median 1-year OCTN2 protein < median

0.8 Median survival (days) 0.8 Median survival (days) *** OCTN2 > median 268 ** OCTN2 > median 254 OCTN2 < median 528 OCTN2 < median 580 0.6 0.6 log-rank P = 0.0045 log-rank P = 0.0008 HR = 2.16 [95% CI 1.25–3.37] HR=2.69 [95% CI 1.47–4.91] 0.4 0.4 Cumulative survival 0.2 Cumulative survival 0.2

0.0 0.0 0 500 1,000 1,500 2,000 0 500 1,000 1,500 2,000 Survival time (days) Survival time (days) 30 26 15 12 5 4 3 2 1 0 0 0 0 0 0 0 0 0 0 0 28 25 12 10 5 2 1 1 0 0 0 0 0 0 0 0 0 0 0 0 30 30 20 20 16 11 7 7 6 6 3 3 3 3 3 3 2 1 0 0 28 25 19 19 14 11 8 8 7 7 4 4 3 3 3 3 1 1 0 0

C Median survival (Days) (Months) (1-YS) (2-YS) D Kaplan-Meier survival analysis: L-carnitine

OCTN2 > median 267.5 8.8 ** 26% 6% 1.0 L-carnitine > median OCTN2 < median 527.5 17.3 64% 23% mRNA L-carnitine < median 0.8 Median survival (days) OCTN2 > median 254.4 8.4 *** 25% 4% L-carnitine > median 381 L-carnitine < median 408 OCTN2 < median 579.5 19.0 63% 30% 0.6 Protein log-rank P = 0.9816 HR = 1.01 [95% CI 0.5–2.04] EF 0.4

100 1,000 0.2 Spearman Spearman Cumulative survival r = −0.007 r = −0.098 P = 0.9706 100 P = 0.6080 n n 10 = 32 = 30 0.0

10 0 500 1,000 1,500 2,000 1 Survival time (days) 1 17 16 11 9 5 5 1 1 0 0 0 0 0 0 0 0 0 0 0 0 17 14 8 7 5 3 3 3 2 2 2 2 1 1 1 1 1 1 0 0 0.1 0.1 OCTN2 mRNA (arb. unit)

0 0.05 0.10 0.15 OCTN2 Protein (arb. unit) 0 0.05 0.10 0.15 L-carnitine L-carnitine

Figure 2. Survival and correlation analyses of OCTN2 and LC in primary GBM. Kaplan–Meier estimates for patients with primary GBM based on their OCTN2 mRNA (A)and protein (B) expression. Patients were divided into 2 subgroups depending on median mRNA or protein expression, log-rank test, , P < 0.01 and , P < 0.001. C, Summary of the survival analysis from A and B showing the median survival time and the 1- and 2-year survival rate (1-YS, 2-YS) for both OCTN2 mRNA and protein expression at a glance. D, Kaplan–Meier estimates for patients with GBM based on their intratumoral LC levels. Patients were divided into 2 subgroups depending on median LC content, log-rank test. E, Correlation between OCTN2 mRNA and intratumoral LC level in primary GBM (Spearman correlation analysis). F, Correlation between OCTN2 protein expression and intratumoral LC level in primary GBM (Spearman correlation analysis).

OCTN2–expressing group [unadjusted HR: 2.69 (95% CI, 1.47– with GBM having a high intratumoral LC level (>median) 4.91, P < 0.001; Fig. 2B)]. Later, the HR for death was adjusted accounted for 381 days versus 408 days for patients with a by fitting the Cox proportional-hazard models. In addition to the lower LC content [ 70 years at diagnosis) with nearly the same group size. compared with 64% with a low intratumoral OCTN2 content. A The ANOVA II analyses revealed no significant interactions similar observation was also made 2 years after diagnosis with between the OCTN2 mRNA or protein expression and age at survival rates of 6% (high OCTN2 expression) versus 30% in the diagnosis (Supplementary Fig. S1E and S1F). However, OCTN2 low OCTN2–expressing group. mRNA and protein expression tended to be lower in female The LC concentration in primary GBM was not associated with patients. In line with the results given in the Cox regression, the survival time (Fig. 2D). The median survival time for patients ANOVA II analysis revealed a significant influence of the OCTN2

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ABC RCTx <50 years 60−70 years <50 years 60−70 years 50−59 years >70 years 50−59 years >70 years Radiomonotherapy

700 800 700

600 600

600 500 500 Figure 3. ANOVA II analysis of OCTN2 mRNA 400 400 and protein expression on patient 400 survival with regard to potential 300 300 confounders. ANOVA II analysis of Survival time (days) Survival time (days) Survival time (days) 200 200 patient survival depending on the 200 OCTN2 mRNA (A) or protein (B) expression in the 4 age classes. 100 * * 100 * 0 OCTN2 expression was divided OCTN2 mRNA OCTN2 mRNA OCTN2 Protein OCTN2 Protein OCTN2 mRNA OCTN2 mRNA according to the median. Inﬂuence > median < median > median < median > median < median of OCTN2 mRNA (C) and protein (E) expression on GBM patient D E F survival depending on the Total resection RCTx Total resection therapeutic regimen (RCTx Subtotal resection Radiomonotherapy Subtotal resection treatment vs. radiomonotherapy). 700 600 600 Inﬂuence of OCTN2 mRNA (D)or 600 protein (F) expression on patient 500 500 survival depending on the resection status of the patients (total vs. 500 400 400 subtotal tumor resection). Tukey- HSD post hoc test (, P < 0.05; 400 , P < 0.01). 300 300 300 Survival time (days) Survival time (days) Survival time (days) 200 200 200

100 * 100 ** 100 **

OCTN2 mRNA OCTN2 mRNA OCTN2 Protein OCTN2 Protein OCTN2 Protein OCTN2 Protein > median < median > median < median > median < median

expression level on survival time of patients with GBM in each GBM, caused a decreased viability of LN18 cells cultured in serum- of the 4 age groups [P ¼ 0.04 (mRNA) and P ¼ 0.01 (protein), starved medium (78%). This effect was reversed in the presence of Fig. 3A and B]. 50 mmol/L LC (109%). Furthermore, the patient survival time for both the RCTx Later, the influence of LC on H2O2-induced oxidative stress and radiomonotherapy group is significantly shorter at high in LN18 cells was evaluated. Coadministration of LC over- OCTN2 levels in the resected GBM samples [vs. low OCTN2 compensated (up to 108%) the viability loss of H2O2-treated expression level, Fig. 3C and E, P ¼ 0.04 (mRNA) and P ¼ LN18 cells (89%) compared with the respective control cells 0.001 (protein)]. A similar effect was observed for the resection (Fig. 4C). To subsequently assess whether the cytoprotective status [Fig. 3D and F, P ¼ 0.04 (mRNA) and P ¼ 0.008 (protein)]. effect of LC depends on its function as a mitochondrial It is noteworthy that in patients with a total tumor resection, the fatty acids shuttle and thus on metabolic effects based on effect of OCTN2 expression is somewhat more pronounced in b-oxidation, the carnitine palmitoyltransferase-1 (CPT-1) comparison with the subtotal resection group. Regarding this inhibitor etomoxir (ETO) (29) was used. Furthermore, the aspect, specific subanalysis via Cox regression revealed an adjust- experiments were carried out under hypoxic conditions in ed HR of 4.6 for death in the high OCTN2 protein–expressing parallel to examine additional hypoxic stress. As shown group with a total tumor resection (95% CI, 1.63–13.12, in Fig. 4D and E, ETO reduced the viability of LN18 cells P ¼ 0.004), and an adjusted HR of 3.3 for patients having received significantly. The effect of ETO was more pronounced under RCTx (95% CI, 1.31–8.37, P ¼ 0.01). hypoxic conditions. Under both normoxic and hypoxic conditions, the cell viability loss was partially reversed by admin- Influence of LC and OCTN2 on survival of LN18 GBM cells istration of 50 mmol/L LC. in vitro In addition, the impact of OCTN2 on GBM cell growth was To investigate the impact of OCTN2 and its substrate LC in studied after siRNA-mediated OCTN2 knockdown. siRNA treat- GBM cells, in vitro experiments using the LN18 GBM cell line were ment resulted in significantly reduced OCTN2 expression and performed. Cells treated with LC had only a slightly increased function as shown by mRNA analysis and 3H-labeled LC uptake viability after 48-hour serum deprivation (116%), which, how- experiments (Fig. 4F and G). Subsequently, we determined the ever, was significantly elevated in a 72-hour period of starvation influence of OCTN2 knockdown on viability of LC-pretreated (142%, Fig. 4A). Furthermore, administration of temozolomide, (50 mmol/L) LN18 cells (Fig. 4H). Under both normoxic and the chemotherapeutic agent used in standard of care treatment of hypoxic conditions knockdown of OCTN2 caused a significant

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OCTN2/LC System in GBM: Cell Protection and Prognostic Value

3 loss of viability of LN18 cells which was, again, more pronounced (2,366 log10[cm ]) compared with the starting time point 3 in cells confronted with hypoxic stress. (2,306 log10[cm ]). In contrast, control animals showed signif- 3 icant progress in tumor size from 2,385 log10[cm ] (treatment 3 Influence of LC on apoptosis and migration of LN18 starting point) to 1,618 log10[cm ] at day 26, which was GBM cells significantly different from the corresponding time point of the To analyze whether LC protects GBM cells from apoptosis meldonium group (P < 0.05). This significant difference was also caused either by cytostatics or oxidative stress, caspase-3 activity present in the growth rate calculations of the tumors (Fig. 5E), 3 was used as a marker for apoptotic processes. LN18 cells treated which showed a mean tumor growth rate of 0,7671 log10[cm ]in 3 with LC showed no significant alterations in caspase-3 activity control animals and 0,06014 log10[cm ] in meldonium-treated (Fig. 5A). In contrast, doxorubicin (7-fold) and carmustine animals (P ¼ 0.01). Three representative MRI recordings for the (BCNU, 1.8-fold) led to a significant increase in caspase-3 activity starting time point (12 days postinjection) and at day 26 post- after 48 hours. Coadministration of LC reduced this effect in injection per group (low, mid, high tumor growth) are shown BCNU-treated cells [1.8-fold (BCNU alone) to 1.2-fold (BCNU þ in Fig. 5F. LC)], but not for doxorubicin. Temozolomide and H2O2 had no effect on caspase-3 activity. Furthermore, cell migration was analyzed using a wound closure assay since migration of GBM Discussion cells into healthy brain is a main feature of this brain tumor. Therapy failure caused by GBM resistance is the most com- Treatment with BCNU and temozolomide did not alter cell mon reason of tumor recurrence resulting in very poor survival migration, whereas administration of ETO, an inhibitor of b-oxi- rates. It has been previously shown that the OCTN2 substrate dation, significantly reduced migration of LN18 cells to about LC acts as a cytoprotector promoting cellular resistance and 50%. Coadministration of LC did not cause any changes in survivability (7, 30, 31). In this study, we demonstrate that migration (Fig. 5B). After the detection of ETO-induced inhibition OCTN2 expression and tissue LC concentrations are signifi- of LN18 cell migration, we assessed expression of 2 key enzymes cantly higher in GBM compared with healthy brain. We of the mitochondrial b-oxidation, CPT1A (carnitine palmitoyl- observed a stepwise increase of OCTN2 expression from transferase 1A) and CPT1C (carnitine palmitoyltransferase 1C), low-grade astrocytoma to primary and recurrent GBM. These in GBM tissue. CPT1A and CPT1C showed a similar expression findings are consistent with those of Bayraktar and colleagues pattern in NMB compared with GBM specimens (Supplementary who described a gradual increase in LC levels and some of its Figs. S2A and S2B). Kaplan–Meier analyses revealed no significant acyl derivates from low- to high-grade astrocytomas through to influence of CPT1A or CPT1C expression on patient survival primary GBM (32). indicating a limited prognostic value in GBM (Supplementary In a recent study, Singer and colleagues described the Figs. S2C and S2D). development of therapeutic resistance in GBM stem cells by enhanced expression of an antioxidant response system referr- Influence of the OCTN2/LC inhibitor meldonium on in vivo ed to as Xc catalytic subunit xCT (SLC7A11) that partially GBM growth operates through activation of the NRF2 transcriptional net- To finally show proof of the concept of the OCTN2/LC system work (19). This network is also addressed by LC (15, 31) and as a therapeutic target for GBM treatment, we analyzed the in vivo regulates antioxidant response element (ARE)-containing tumor growth in an orthotopic murine GBM model using the cell genes (33) leading to attenuation of ROS-mediated cell dam- line GL261, which was stereotactically injected into mice brain. age, arrest of apoptosis, and resistance to radiochemother- Twelve days postinjection, tumor development was assessed by apy (31, 34). ROS homeostasis is strictly regulated by cancer MRI. Afterwards, mice were treated with either 0.9% NaCl (con- cells to promote tumorigenesis and malignant transforma- trol animals) or 250 mg/kg meldonium as an OCTN2/LC inhib- tion (17, 35, 36). OCTN2 upregulation and LC accumulation itor (28) every day intraperitoneally until day 26 postinjection. could therefore help GBM cells to maintain conducive ROS To ensure normality of data, tumor volumes were log-trans- levels both to drive mutagenesis and to mitigate unfavorable formed. Also, tumor growth was calculated as the difference conditions evoked by ROS-generating processes, for example, between the transformed volumes. As seen in Fig. 5C–E at day high metabolic turnover and radiochemotherapy (19, 20). 26 postinjection (14 days after starting treatment), a decrease in These considerations are underlined by our findings that LN18 mean tumor size was observed in the meldonium-treated group GBM cells, a well-accepted WHO grade IV GBM cell line (37),

(Continued.) Cell viability was normalized to solvent (DMSO þ aqua dest.) treated control cells (100%; 1-way ANOVA; , P < 0.05; , P < 0.001 vs. solvent, # , P < 0.01 TMZ þ LC vs. 100 mmol/L TMZ alone). C, Influence of LC on H2O2-induced loss of LN18 cell viability. Cells were preincubated for 24 hours with 50 mmol/L LC followed by application of H2O2 (10, 50, or 100 mmol/L) either alone or in combination with 50 mmol/L LC for 30 minutes in SSM. After further # ## 24 hours, the cell viability was determined, 1-way ANOVA ( , P < 0.05; , P < 0.001 vs. solvent (aqua dest.); , P < 0.05; , P < 0.01 H2O2 þ LC vs. H2O2 alone). Impact of etomoxir (ETO) on LN18 cell viability under normoxia (D, 21 Vol.-% O2) and hypoxia (E, 0.5 Vol.-% O2). Cells were pretreated for 24 hours with 50 mmol/ L LC followed by application of 100 mmol/L ETO either alone or in combination with 50 mmol/L LC. After 48, 72, or 96 hours, cell viability was measured [1-way ANOVA; , P < 0.001 vs. aqua dest. (solvent); ##, P < 0.01; ###, P < 0.001 ETO þ LC vs. ETO alone]. F–H, siRNA-mediated knockdown of OCTN2 expression in LN18 cells. F, OCTN2 mRNA expression 48 and 72 hours after transfection with either scrambled control siRNA or OCTN2-specific siRNA (unpaired t test, , P < 0.001 vs. control siRNA). G, LC uptake into LN18 cells transfected with either scrambled control siRNA or OCTN2-specific siRNA. Cells were incubated with 100 nmol/L 3H-labeled LC at 37C for 30 minutes (unpaired t test , P < 0.01 vs. control siRNA). H, Determination of cell viability using resazurine assay of

LC-treated (50 mmol/L) LN18 cells 48 or 72 hours after transfection with either scrambled control siRNA or OCTN2-speciﬁc siRNA under normoxia (21 Vol.-% O2) and hypoxia (0.5 Vol.-% O2). Cell viability was normalized to cells transfected with scrambled control siRNA (100%; 1-way ANOVA , P < 0.05; , P < 0.001 vs. control siRNA; #, P < 0.05 normoxia vs. hypoxia). All data are shown as mean SD (n ¼ 3; each in triplicates; arb., arbitrary).

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AB0 µmol/L LC Control 250 µmol/L BCNU 50 µmol/L LC 100 µmol/L ETO 250 µmol/L TMZ 10 100 ** ** 8 80

60 6 ## ** ** ** ** 2 40 ** (arb. unit)

1 20 Wound closure (%) Relative caspase-3 activity 0 0 DMSO Doxo TMZ BCNU H O H O 0 0 0 0 2 2 2 50 50 50 50 Control 1 μmol/L 100 μmol/L 100 μmol/L Control 200 μmol/L 100 500 100 500 100 500 100 500 LC [μmol/L]

CDEControl Control Control Meldonium Meldonium Meldonium 0 0 2 * )

-1 -1 1 1 V ) ) ) 3 3 3 log( (cm (cm (cm -2 -2 * - 0 ) 10 10 10 2 V og log l log

-3 -3 log( -1 Log tumor volume Log tumor volume n = 10 n = 9 -4 -4 -2 d12 d26 d12 d26 Control Meldonium

F Control Meldonium d12 d26 d12 d26 Low tumor growth Mid tumor growth High tumor growth

Figure 5. Inﬂuence of LC on caspase-3 activity and migration of LN18 GBM cells as well as analysis of OCTN2 as a therapeutic target in an orthotopic in vivo mouse model. A, Analysis of caspase-3 activity in LN18 cells after treatment with doxorubicin (Doxo, 1 mmol/L), temozolomide (TMZ, 100 mmol/L), carmustine (BCNU, 100

mmol/L), H2O2 (200 mmol/L), or solvent (DMSO for doxorubicin, TMZ, and BCNU; H2OforH2O2) either alone or in combination with LC (50 mmol/L). Cells were treated with the indicated substances for 48 hours in serum-starved medium (SSM, 0.05% FCS). Caspase-3 activity was normalized to solvent-treated control cells, 2-way ANOVA with Bonferroni post hoc test (, P < 0.01 vs. control cells; ##, P < 0.05 for with vs. without LC (arb., arbitrary). B, Wound closure assay for determination of the migratory capacity of LN18 cells after stimulation with etomoxir (ETO, 100 mmol/L), BCNU (250 mmol/L), temozolomide (250 mmol/L), and DMSO (control) either alone or in combination with LC (50, 100, and 500 mmol/L). The migratory capacity is depicted as wound closure in percent after an incubation time of 24 hours [2-way ANOVA with Bonferroni post hoc test; , P < 0.001 vs. control cells (without any drugs), all data are shown as mean SD]. C and D, Inﬂuence of the OCTN2 inhibitor meldonium on in vivo GBM growth in an orthotopic mouse model using murine GL261 GBM cells. Mice were intraperitoneally treated from day 12 to 26 postinjection every day with either 0.9% NaCl (control animals, n ¼ 10) or 250 mg/kg meldonium (n ¼ 9) as OCTN2 inhibitor. Tumor volume was calculated with Horos software in coronal and axial gadolinium-enhanced T1-weighted sections 12 days and 26 days postinjection; data were log-transformed. C shows individual animals while D shows the mean SD, unpaired t test, , P < 0.05. E, Comparison of tumor growth rates in control

group versus meldonium-treated group. Growth rate was calculated as difference between the log-transformed tumor volumes on day 26 (V2) and day 12 (V1). Positive values indicate growth while negative values indicate downsizing, unpaired t test, , P < 0.05. F, Representative gadolinium-enhanced T1-weighted images (coronal) for animals with low, mid, and high tumor growth from each group (control: 0.9% NaCl; meldonium: 250 mg/kg) at the ﬁrst day of pharmacologic intervention (day 12 postinjection) and at the end of the study (day 26 postinjection).

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OCTN2/LC System in GBM: Cell Protection and Prognostic Value

become more resistant or at least maintain their viability under To our knowledge, no association studies on the role of the hypoxic, metabolic, and cytotoxic stress when pretreated with LC. OCTN2/LC system in GBM survival have been published to date. The results are consistent with several in vitro and in vivo studies Here, after adjustment of the HR on therapeutic regimen, resec- on LC's antioxidant and antiapoptotic activities, which have tion status, age, and sex, we found a high OCTN2 protein been predominantly studied in nonmalignant compromised expression to correlate with a 2.9-fold increased risk for death in cells in context of neurodegenerative disorders (10, 11), but are patients with primary GBM. However, it has to be mentioned that also detectable in the malignant neuroblastoma cell line further potential confounders (e.g., ECOG Performance Status, SH-SY5Y (12). However, LC enhanced the viability of the human Karnofsky Index) were not recorded. The ANOVA II analyses LN18 GBM cells, but had no detectable effect on cell migration. revealed that differences in the patient survival between the low Following administration of LC, we also observed a decreased and high OCTN2–expressing group were highly pronounced in caspase-3 activity in LN18 cells treated with BCNU and a restored the respective subpopulation receiving RCTx and a total tumor viability in cells treated with temozolomide, both drugs used in resection, suggesting a prognostic relevance especially in holisti- the therapeutic regimen for patients with GBM. These results cally treated patients. endorse the theory that LC strengthens the tumor cells through Although OCTN2 expression has been described in various antiapoptotic mechanisms or perhaps through a combination of tumor cells (43–45), its precise role in malignant transforma- several cytoprotective effects. However, the cytoprotective LC tion remains elusive. Contrary to our observations, Scalise concentrations given in literature are widely spread within a range and colleagues found a downregulation of OCTN2 expression of 9–25 mmol/L (38), 30–100 mmol/L (39), 0.1–1 mmol/L (40), in epithelial cancer cell lines, whereas an estrogen-mediated and 1–10 mmol/L (41), respectively. In our current work, a LC induction of OCTN2 expression in breast cancer was concentration of 50 mmol/L was found to be adequate in detecting described (43, 44). For GBM, however, our data of increased cytoprotective effects. Lower LC concentrations showed no OCTN2 expression may provide both a prognostic factor for significant benefit in our in vitro experiments while higher LC GBM treatment as well as an option for optimizing GBM concentrations did not increase the survival-promoting effects therapybyeitherspecific blocking of OCTN2 or drug targeting (data not shown) even though other studies claim a concentration via OCTN2. Very recently, it was shown that LC-conjugated dependent effect of LC (6, 12, 30). Of note, the LC concentration nanoparticles promote permeation across the blood–brain used in our cell experiments was within the physiologic range of barrier to target glioma cells via OCTN2 resulting in improved the LC plasma concentration, which is generally estimated to be antiglioma therapy (46). Our preclinical studies using an 40–50 mmol/L (42). orthotopic GBM mouse model demonstrate significantly Recent NMR analyses of in situ glioma specimens as well as reduced intracerebral tumor growth through inhibition of the further in vitro and in vivo studies suggest that oxidative metab- OCTN2/LC system and suggest it as a potential target in GBM olism plays an integral role of cellular maintenance and pro- therapy. To date, our in vivo study is the first to show the liferation in malignant glioma cells (5). Because LC is crucial antitumoral efficacy of the OCTN2/LC inhibitor meldonium. for transporting middle- and long-chain fatty acids into mito- While no major side effects of meldonium have been observed chondria to drive b-oxidation, some consideration had to be so far, meldonium has even been shown to improve cardio- given to how LC's metabolic effects could influence the tumor vascular function (47, 48). cells' behavior. We used the irreversible CPT1 inhibitor eto- Some limitations of our study need to be addressed. Our moxir (ETO) to block b-oxidation to analyze whether GBM work represents a global expression analysis of the entire tumor cells rely on fatty acid oxidation and to what extent cytoprotec- mass without tumor region or cell type–specific differentiation, tion is attributable to LC when uncoupled from its native especially considering the heterogeneity of GBM. Taking into function as a mitochondrial shuttle. ETO caused both a sig- account the different types of GBM and their distinct genetic nificant loss of cell viability and migration in LN18 cells, which and epigenetic profiles (49), predictive molecular biomarkers has been also shown in primary GBM cells (5, 29). Under both for therapy response and outcome may vary in respective GBM normoxic and hypoxic cell culture conditions, the loss of subtypes. Our patient cohort was not subclassified and we viability, but not the ETO-induced inhibition of migration, therefore cannot draw any conclusion regarding differences in was abolished by simultaneous LC application. These results GBM subtypes. In our study, only vital tumor samples from the provide 3 valuable hints: First, LN18 relies to a remarkable edge region were used. Within the same tumor, hypoxic, extent on fatty acid metabolism as bioenergetic fuel and lacks necrotic, and highly vascularized areas vary in their expression viability and migration when b-oxidation is blocked. Second, profiles and thus also in their OCTN2 expression. In addition, LC stabilizes cell integrity independently of its metabolic func- the use of only 1 cell line for in vitro and in vivo studies is a tion, but rather by its abovementioned extramitochondrial limitation of our investigations, arguing toward analyses in cytoprotective effects (31, 34). Third, LC did not reverse or further human cell lines or primary GBM cells to validate the alleviate the ETO-induced inhibition of migration suggesting observed effects. that LC has no perceptible influence on GBM cell migration Nevertheless, this study stresses the role of OCTN2 and its based on metabolic effects. It is quite possible, however, that substrate LC in GBM cytoprotection, suggests OCTN2 as a malignant GBM cells gain survival advantages on both cyto- prognostic marker and offers a novel target that may aid in protective and metabolic levels through OCTN2 overexpression combatting tumor resistance or increasing its sensitivity to and LC accumulation, as it is also assumed that fatty acid therapy. Although the precise mechanisms of the OCTN2/LC oxidation provides NADPH for defense against oxidative stress system in GBM biology remain to be completely determined, and cell death in GBM cells (29). Nevertheless, the molecular the promising results of meldonium in reducing tumor growth details of malignant transformation and resistance of tumor provide strong support for further research on the OCTN2/LC cells need to be further elaborated in this context. system, which is aimed at improving antiglioma therapy.

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Disclosure of Potential Conﬂicts of Interest Administrative, technical, or material support (i.e., reporting or organizing € No potential conﬂicts of interest were disclosed. data, constructing databases): M.A. Fink, H. Paland, S. Bien-Moller Study supervision: M.A. Fink, H.W.S. Schroeder, S. Bien-Moller€

Authors' Contributions Acknowledgments Conception and design: M.A. Fink, H. Paland, S. Bien-Moller€ This work was supported by national funding from the Forschungsverbund Development of methodology: M.A. Fink, H. Paland Neurowissenschaften. M.A. Fink received a scholarship of the Gerhard Domagk Acquisition of data (provided animals, acquired and managed Program, funded by the Faculty of Medicine, Greifswald, Germany, and was patients, provided facilities, etc.): M.A. Fink, H. Paland, S. Herzog, further supported by the Germany Scholarship, funded by the German Federal M.Grube,S.Vogelgesang,A.Bialke, W. Hoffmann, H.W.S. Schroeder, Ministry for Education and Research. S. Bien-Moller€ Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The costs of publication of this article were defrayed in part by the payment computational analysis): M.A. Fink, H. Paland, K. Weitmann, W. Hoffmann, of page charges. This article must therefore be hereby marked advertisement B.H. Rauch, S. Bien-Moller€ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Writing, review, and/or revision of the manuscript: M.A. Fink, H. Paland, M. Grube, S. Vogelgesang, K. Weitmann, A. Bialke, W. Hoffmann, B.H. Rauch, Received July 24, 2018; revised December 4, 2018; accepted January 10, 2019; S. Bien-Moller€ published ﬁrst January 22, 2019.

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OCTN2/LC System in GBM: Cell Protection and Prognostic Value

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www.aacrjournals.org Clin Cancer Res; 2019 OF13

L-Carnitine−Mediated Tumor Cell Protection and Poor Patient Survival Associated with OCTN2 Overexpression in Glioblastoma Multiforme

Matthias A. Fink, Heiko Paland, Susann Herzog, et al.

Clin Cancer Res Published OnlineFirst January 22, 2019.

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