Fatty acid oxidation is required for the respiration and proliferation of malignant glioma cells

Abstract

Background: Glioma is the most common form of primary malignant brain tumor in adults, with approximately 4 cases per 100 000 people each year. Gliomas, like many tumors, are thought to primarily metabolize glucose for energy production; however, the reliance upon glycolysis has recently been called into question. In this study, we aimed to identify the metabolic fuel requirements of human glioma cells.

Methods: We used database searches and tissue culture resources to evaluate genotype and protein expression, tracked oxygen consumption rates to study metabolic responses to various substrates, performed histochemical techniques and fluorescence-activated cell sorting-based mitotic profiling to study cellular proliferation rates, and employed an animal model of malignant glioma to evaluate a new therapeutic intervention.

Results: We observed the presence of enzymes required for fatty acid oxidation within human glioma tissues. In addition, we demonstrated that this metabolic pathway is a major contributor to aerobic respiration in primary-cultured cells isolated from human glioma and grown under serum-free conditions. Moreover, inhibiting fatty acid oxidation reduces proliferative activity in these primary-cultured cells and prolongs survival in a syngeneic mouse model of malignant glioma.

Conclusions: Fatty acid oxidation enzymes are present and active within glioma tissues. Targeting this metabolic pathway reduces energy production and cellular proliferation in glioma cells. The drug etomoxir may provide therapeutic benefit to patients with malignant glioma. In addition, the expression of fatty acid oxidation enzymes may provide prognostic indicators for clinical practice.

Keywords: etomoxir; fatty acid oxidation; glioblastoma; glioma; metabolism.

Fig. 1

Human glioblastomas express enzymes required for fatty acid oxidation Representative photomicrographs demonstrate staining in human glioblastoma tissue for medium-chain acyl-CoA dehydrogenase (MCAD, A), short-chain hydroxyacyl CoA dehydrogenase (SCHAD, B), very-long-chain acyl-CoA dehydrogenase (VLCAD, C), carnitine palmitoyl transferase 1a (CPT1a, D), and the trifunctional protein hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (HADHA, E). The majority of mitochondria labeled with the pan-mitochondrial marker succinate dehydrogenase (SDHA) are co-labeled with HADHA (F-J). A fraction of cells in human glioblastoma tissue identified with Hoechst (K,O,S) and expressing MCAD (L,P,T) co-label with the astroglial marker GFAP (M), the neural progenitor marker SOX2 (Q), and the glial progenitor marker OLIG2 (U). Representative abeled cells are shown in merged images (N,R,V). The fraction of cells expressing each of the fatty acid oxidation enzymes is shown (W), as well as the fraction of total cells (X) and MCAD+ cells (Y) co-labeled with each cell type. Scale bars are 10 µm.

Fig. 2

Fatty acid oxidation is a primary contributor to aerobic respiration in primary-cultured hGBMs. The oxygen consumption rate (OCR) of primary-cultured serum-free hGBM cells was assessed using the Seahorse Analyzer (A). Baseline measurements were taken for cells in plain medium (empty circles), with glutamine (black triangles), with glucose (black squares), and exposed to 10% FBS for 72 hours prior to the experiment (grey diamonds). Cells were then treated with 100 µM linoleic acid (a polyunsaturated fatty acid), 100 µM etomoxir (an inhibitor of beta-oxidation), 2.0 µM FCCP (which induces maximal respiration), and 2.5 µM antimycin A (which inhibits aerobic respiration). Spare respiratory capacity, calculated by dividing basal OCR by maximal OCR, is shown (B). Cellular response to linoleic acid (light grey bars) and etomoxir (dark grey bars) is shown (C). The fraction of mitochondrial respiration dependent on fatty acids is shown (D). A similar experiment was conducted to evaluate responses to glucose and the glycolytic inhibitor 2-DG (E). Cellular responses are summarized (F), and the fraction of mitochondrial respiration dependent on glucose oxidation or glycolysis is shown (G). *indicates significant change in respiration, P < .05.

Fig. 3

Inhibition of fatty acid oxidation decreases proliferation in hGBMs but does not affect cellular survival. Sample photomicrographs of cells treated with phosphate-buffered saline (PBS) (A, D), 100 µM etomoxir (B, E), or 100 µM linoleic acid (C, F) are shown, stained with either TdT, a marker of apoptosis (A–C), or KI67, an S-phase cell cycle marker (D–F). Separate channels are shown to display Hoechst, a pan-nuclear marker ('), and TdT or KI67 (''). The fraction of TdT+ apoptotic cells did not change significantly in either treatment group (P > .05, G). The fraction of KI67+ proliferating cells decreased upon 24 hours treatment with etomoxir (P < .05, H) and increased upon 24 hours of treatment with linoleic acid (P < .01, H). The total cell count after 24 hours was significantly decreased in the presence of etomoxir (P < .01, I) but was unaffected in the presence of linoleic acid (P > .05, I). Shown are representative data from fluorescence-activated cell sorting-based analysis of the mitotic index of primary-cultured serum-free hGBM cells, performed after treatment with PBS (J), 100 µM etomoxir (K), or 100 µM linoleic acid (L). The fraction of cells in S + G2/M phase of the cell cycle decreased by 20% from control levels upon treatment with 100 µM etomoxir (P < .01, M); this fraction was unaffected by treatment with linoleic acid (P > .05, M).

Fig. 4

Inhibition of fatty acid oxidation by etomoxir prolongs survival time in a syngeneic mouse model of malignant glioma. Fourteen days after surgical implantation of glioma-initiating cells into the brains of wild-type adult mice, osmotic pumps were subcutaneously implanted to deliver drug or control substance for a period of 30 days in a blinded, placebo-controlled preclinical study (A). Animals were monitored until euthanasia criteria were met. Weight loss and other symptoms occurred primarily in the final 3 days of life (B) and were similar between the control and treatment groups (8.0 g vs 7.5 g, respectively, P > .05). Mice treated with 10 mg/kg etomoxir sodium salt lived significantly longer than animals treated with vehicle control (C, P < .001, ANOVA).

Fig. 5

No difference in tumor size, location, or phenotype was observed between treatment groups at clinical endpoint. Brains from the mice in the study were photographed to evaluate tumor severity. (A) No significant difference in tumor volume or macro score was observed between treatment groups at clinical endpoint (B–C), corresponding to similar tumor grading by histological assessment (D). Infiltration of tumors from the injection site (striatum, E) into corpus collosum (F), cortex (G), dorsal aspects of the brain (H), across midline (I), or into hippocampus (J) were not found to be significantly different between treatment groups (P > .05, chi-square test). Tumors were further evaluated to quantify KI67+ proliferation index (K, N), p53+ malignancy marker (L, O), and GFAP+ glial cell content (M, P) in control-treated animals (K-M) and etomoxir-treated animals (N–P). No difference in cellular phenotype was observed between groups (P > .05, 2-tailed t test).

Fig. 6

Emergence and progression of glioma were delayed upon treatment with the investigational drug etomoxir. Mouse 077 (control) and mouse 078 (etomoxir) were subjected to MRI to track tumor progression during the study. Coronal images throughout the brain are shown for Mouse 077 across the entire experimental time-course (A-E). At 46 days post cell implantation (DPI), an injection track was observed with no evidence of tumor (A). At 53 DPI, there was no evidence of tumor and no clinical symptoms (B). At 67 DPI, a 1.0 mm tumor was observed with no clinical symptoms (C). At 72 DPI, a 1.3 mm tumor was observed with approximately 5% loss of body weight the following day (D). At 75 DPI, a 5 mm tumor was observed, with approximately 15% loss of body weight and other symptoms signaling clinical endpoint (E). A zoom image of this time point is shown (F). Tumor growth was slowed upon treatment with 10 mg/kg/day etomoxir in mouse 078 (G-K). This animal first manifested tumor by MR at 95 DPI; at 97 DPI, this animal lost 15% body weight and reached clinical endpoint. A zoom image of this animal's brain at 75 DPI is shown to compare with the vehicle-treated animal at the same time point (L).