RESUMEN
Knockout (KO) of the fatty acid-activation enzyme very long-chain acyl-CoA synthetase 3 (ACSVL3; SLC27A3) in U87MG glioblastoma cells reduced their malignant growth properties both in vitro and in xenografts. These U87-KO glioma cells grew at a slower rate, became adherence-dependent, and were less invasive than parental U87 cells. U87-KO cells produced fewer, slower-growing subcutaneous and intracranial tumors when implanted in NOD-SCID mice. Thus, depleting U87MG cells of ACSVL3 restored these cells to a phenotype more like that of normal astrocytes. To understand the mechanisms underlying these beneficial changes, we investigated several possibilities, including the effects of ACSVL3 depletion on carbohydrate metabolism. Proteomic and metabolomic profiling indicated that ACSVL3 KO produced changes in glucose and energy metabolism. Even though protein levels of glucose transporters GLUT1 and GLUT3 were reduced by KO, cellular uptake of labeled 2-deoxyglucose was unaffected. Glucose oxidation to CO2 was reduced nearly 7-fold by ACSVL3 depletion, and the cellular glucose level was 25% higher in KO cells. Glycolytic enzymes were upregulated by KO, but metabolic intermediates were essentially unchanged. Surprisingly, lactate production and the levels of lactate dehydrogenase isozymes LDHA and LDHB were elevated by ACSVL3 KO. The activity of the pentose phosphate pathway was found to be lower in KO cells. Citric acid cycle enzymes, electron transport chain complexes, and ATP synthase protein levels were all reduced by ACSVL3 depletion. Mitochondria were elongated in KO cells, but had a more punctate morphology in U87 cells. The mitochondrial potential was unaffected by lack of ACSVL3. We conclude that the beneficial effects of ACSVL3 depletion in human glioblastoma cells may result in part from alterations in diverse metabolic processes that are not directly related to role(s) of this enzyme in fatty acid and/or lipid metabolism. (Supported by NIH 5R01NS062043 and KKI institutional funds.).
RESUMEN
Decreasing the expression of very long-chain acyl-CoA synthetase 3 (ACSVL3) in U87MG glioblastoma cells by either RNA interference or genomic knockout (KO) significantly decreased their growth rate in culture, as well as their ability to form rapidly growing tumors in mice. U87-KO cells grew at a 9-fold slower rate than U87MG cells. When injected subcutaneously in nude mice, the tumor initiation frequency of U87-KO cells was 70% of that of U87MG cells, and the average growth rate of tumors that did form was decreased by 9-fold. Two hypotheses to explain the decreased growth rate of KO cells were investigated. Lack of ACSVL3 could reduce cell growth either by increasing apoptosis, or via effects on the cell cycle. We examined intrinsic, extrinsic, and caspase-independent apoptosis pathways; none were affected by lack of ACSVL3. However, significant differences in the cell cycle were seen in KO cells, suggesting arrest in S-phase. Levels of cyclin-dependent kinases 1, 2, and 4 were elevated in U87-KO cells, as were regulatory proteins p21 and p53 that promote cell cycle arrest. In contrast, lack of ACSVL3 reduced the level of the inhibitory regulatory protein p27. γ-H2AX, a marker of DNA double strand breaks, was elevated in U87-KO cells, while pH3, a mitotic index marker, was reduced. Previously reported alterations in sphingolipid metabolism in ACSVL3-depleted U87 cells may explain the effect of KO on cell cycle. These studies reinforce the notion that ACSVL3 is a promising therapeutic target in glioblastoma.
RESUMEN
Gliomas are the largest category of primary malignant brain tumors in adults, and glioblastomas account for nearly half of malignant gliomas. Glioblastomas are notoriously aggressive and drug-resistant, with a very poor 5 year survival rate of about 5%. New approaches to treatment are thus urgently needed. We previously identified an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), as a potential therapeutic target in glioblastoma. Using the glioblastoma cell line U87MG, we created a cell line with genomic deletion of ACSVL3 (U87-KO) and investigated potential mechanisms to explain how this enzyme supports the malignant properties of glioblastoma cells. Compared to U87MG cells, U87-KO cells grew slower and assumed a more normal morphology. They produced fewer, and far smaller, subcutaneous xenografts in nude mice. Acyl-CoA synthetases, including ACSVL3, convert fatty acids to their acyl-CoA derivatives, allowing participation in diverse downstream lipid pathways. We examined the effect of ACSVL3 depletion on several such pathways. Fatty acid degradation for energy production was not affected in U87-KO cells. Fatty acid synthesis, and incorporation of de novo synthesized fatty acids into membrane phospholipids needed for rapid tumor cell growth, was not significantly affected by lack of ACSVL3. In contrast, U87-KO cells exhibited evidence of altered sphingolipid metabolism. Levels of ceramides containing 18-22 carbon fatty acids were significantly lower in U87-KO cells. This paralleled the fatty acid substrate specificity profile of ACSVL3. The rate of incorporation of stearate, an 18-carbon saturated fatty acid, into ceramides was reduced in U87-KO cells, and proteomics revealed lower abundance of ceramide synthesis pathway enzymes. Sphingolipids, including gangliosides, are functional constituents of lipid rafts, membrane microdomains thought to be organizing centers for receptor-mediated signaling. Both raft morphology and ganglioside composition were altered by deficiency of ACSVL3. Finally, levels of sphingosine-1-phosphate, a sphingolipid signaling molecule, were reduced in U87-KO cells. We conclude that ACSVL3 supports the malignant behavior of U87MG cells, at least in part, by altering cellular sphingolipid metabolism.
