Very long-chain acyl-CoA synthetase 3 mediates onco-sphingolipid metabolism in malignant glioma

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Elizabeth A. Kolar Xiaohai Shi Emily M. Clay Ann B. Moser Bachchu Lal Raja Sekhar Nirujogi Akhilesh Pandey Veera Venkata Ratnam Bandaru John Laterra Zhengtong Pei Paul A. Watkins


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.

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KOLAR, Elizabeth A. et al. Very long-chain acyl-CoA synthetase 3 mediates onco-sphingolipid metabolism in malignant glioma. Medical Research Archives, [S.l.], v. 9, n. 5, may 2021. ISSN 2375-1924. Available at: <>. Date accessed: 28 oct. 2021. doi:
Research Articles


1. Ostrom QT, Patil N, Cioffi G, Waite K, Kruchko C, Barnholtz-Sloan JS. CBTRUS statistical report: Primary brain and other central nervous system tumors diagnosed in the United States in 2013-2017. Neuro Oncol. 2020 Oct 30;22(12 Suppl 2):iv1-iv96. doi:10.1093/neuonc/noaa200
2. Weller M, van den Bent M, Hopkins K, et al. EANO guideline for the diagnosis and treatment of anaplastic gliomas and glioblastoma. Lancet Oncol. 2014;15(9):e395-403. doi:10.1016/S1470-2045(14)70011-7
3. Oronsky B, Reid TR, Oronsky A, Sandhu N, Knox SJ. A Review of Newly Diagnosed Glioblastoma. Front Oncol. Published online 2021. Feb 5;10:574012 doi:10.3389/fonc.2020.574012
4. Pei Z, Sun P, Huang P, Lal B, Laterra J, Watkins PA. Acyl-CoA synthetase VL3 knockdown inhibits human glioma cell proliferation and tumorigenicity. Cancer Res. 2009;69(24). doi:10.1158/0008-5472.CAN-08-4689
5. Bowman RL, Wang Q, Carro A, Verhaak RGW, Squatrito M. GlioVis data portal for visualization and analysis of brain tumor expression datasets. Neuro Oncol. 2017. Jan;19(1):139-141. doi:10.1093/neuonc/now247
6. Watkins PA, Maiguel D, Jia Z, Pevsner J. Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome. J Lipid Res. 2007;48(12). doi:10.1194/jlr.M700378-JLR200
7. Jia Z, Moulson CL, Pei Z, Miner JH, Watkins PA. Fatty acid transport protein 4 is the principal very long chain fatty acyl-CoA synthetase in skin fibroblasts. J Biol Chem. 2007;282(28). doi:10.1074/jbc.M700568200
8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
9. Pei Z, Fraisl P, Berger J, Jia Z, Forss-Petter S, Watkins PA. Mouse very long-chain Acyl-CoA synthetase 3/fatty acid transport protein 3 catalyzes fatty acid activation but not fatty acid transport in MA-10 cells. J Biol Chem. 2004;279(52). doi:10.1074/jbc.M410091200
10. Laterra J, Rosen E, Nam M, Ranganathan S, Fielding K, Johnston P. Scatter factor/hepatocyte growth factor expression enhances human glioblastoma tumorigenicity and growth. Biochem Biophys Res Commun. 1997;235(3):743-747. doi: 10.1006/bbrc.1997.6853.
11. Dole VP. A relation between non-esterified fatty acids in plasma and the metabolism of glucose. J Clin Invest. 1956;35:150-154.
12. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:457-509.
13. Bandaru VVR, Patel N, Ewaleifoh O, Haughey NJ. A failure to normalize biochemical and metabolic insults during morphine withdrawal disrupts synaptic repair in mice transgenic for HIV-gp120. J Neuroimmune Pharmacol. 2011. Dec;6(4):640-9. doi:10.1007/s11481-011-9289-0.
14. McFadden JW, Aja S, Li Q, et al. Increasing fatty acid oxidation remodels the hypothalamic neurometabolome to mitigate stress and inflammation. PLoS One. 2014. Dec 26;9(12):e115642. doi:10.1371/journal.pone.0115642
15. Bandaru VVR, Troncoso J, Wheeler D, et al. ApoE4 disrupts sterol and sphingolipid metabolism in Alzheimer’s but not normal brain. Neurobiol Aging. 2009. Apr;30(4):591-9. doi:10.1016/j.neurobiolaging.2007.07.024
16. Shaner RL, Allegood JC, Park H, et al. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers. J Lipid Res. 2009. Aug;50(8):1692-707. doi:10.1194/jlr.D800051-JLR200
17. Sullards MC, Merrill AH. Analysis of sphingosine 1-phosphate, ceramides, and other bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Sci STKE. 2001. Jan 30;2001(67):pl1. doi:10.1126/stke.2001.67.pl1
18. Petrache I, Natarajan V, Zhen L, et al. Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice. Nat Med. 2005. May;11(5):491-8. doi:10.1038/nm1238
19. Schnaar RL, Needham LK. Thin-layer chromatography of glycosphingolipids. Methods Enzym. 1994;230:371-389. doi: 10.1016/0076-6879(94)30025-9.
20. Nirujogi RS, Wright JD, Manda SS, et al. Phosphoproteomic analysis reveals compensatory effects in the piriform cortex of VX nerve agent exposed rats. Proteomics. 2015. Jan;15(2-3):487-99. doi:10.1002/pmic.201400371
21. Kim MS, Pinto SM, Getnet D, et al. A draft map of the human proteome. Nature. 2014. May 29;509(7502):575-81. doi:10.1038/nature13302
22. Weimer PJ. End product yields from the extraruminal fermentation of various polysaccharide, protein and nucleic acid components of biofuels feedstocks. Bioresour Technol. 2010;102(3):3254-3259. doi:S0960-8524(10)01852-3 [pii]10.1016/j.biortech.2010.11.050
23. Zou Z, DiRusso CC, Ctrnacta V, Black PN. Fatty acid transport in Saccharomyces cerevisiae. Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p. J Biol Chem. 2002;277(34):31062-31071. doi: 10.1074/jbc.M205034200. Epub 2002 Jun 6. PMID: 12052836.
24. Röhrig F, Schulze A. The multifaceted roles of fatty acid synthesis in cancer. Nat Rev Cancer. 2016. Nov;16(11):732-749. doi:10.1038/nrc.2016.89
25. Mollinedo F, Gajate C. Lipid rafts as major platforms for signaling regulation in cancer. Adv Biol Regul. 2015. Jan;57:130-46. doi:10.1016/j.jbior.2014.10.003
26. Villar VAM, Cuevas S, Zheng X, Jose PA. Localization and signaling of GPCRs in lipid rafts. Methods Cell Biol. 2016;132:3-23. doi:10.1016/bs.mcb.2015.11.008
27. Lingwood D, Simons K. Lipid rafts as a membrane-organizing principle. Science (80- ). 2010. Jan 1;327(5961):46-50. doi:10.1126/science.1174621
28. Simons K, Ikonen E. Functional rafts in cell membranes. Nature. 1997. Jun 5;387(6633):569-72. doi:10.1038/42408
29. Strickland M, Stoll EA. Metabolic reprogramming in glioma. Front Cell Dev Biol. 2017. Apr 26;5:43. doi:10.3389/fcell.2017.00043
30. Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 Mutations in Gliomas . N Engl J Med. 2009. Feb 19;360(8):765-73. doi:10.1056/nejmoa0808710
31. Clark O, Yen K, Mellinghoff IK. Molecular pathways: Isocitrate dehydrogenase mutations in cancer. Clin Cancer Res. Published online 2016. doi:10.1158/1078-0432.CCR-13-1333
32. Watkins PA. Fatty acid activation. Prog Lipid Res. 1997;36(1). doi:10.1016/S0163-7827(97)00004-0
33. Pei Z, Fraisl P, Shi X, et al. Very Long-Chain Acyl-CoA Synthetase 3: Overexpression and Growth Dependence in Lung Cancer. PLoS One. 2013;8(7). doi:10.1371/journal.pone.0069392
34. Liu Q, Luo Q, Halim A, Song G. Targeting lipid metabolism of cancer cells: A promising therapeutic strategy for cancer. Cancer Lett. 2017. Aug 10;401:39-45. doi:10.1016/j.canlet.2017.05.002
35. Wakamiya T, Suzuki SO, Hamasaki H, et al. Elevated expression of fatty acid synthase and nuclear localization of carnitine palmitoyltransferase 1C are common among human gliomas. Neuropathology. 2014. Oct;34(5):465-74. doi:10.1111/neup.12132
36. Zhang A, Long W, Guo Z, Guo Z, Cao BB. Downregulation of hepatoma-derived growth factor suppresses the malignant phenotype of U87 human glioma cells. Oncol Rep. 2012. Jul;28(1):62-8. doi:10.3892/or.2012.1768
37. Huang W, Ding X, Ye H, Wang J, Shao J, Huang T. Hypoxia enhances the migration and invasion of human glioblastoma U87 cells through PI3K/Akt/mTOR/HIF-1α pathway. Neuroreport. 2018. Dec 12;29(18):1578-1585. doi:10.1097/WNR.0000000000001156
38. Van Brocklyn JR, Letterle CA, Snyder PJ, Prior TW. Sphingosine-1-phosphate stimulates human glioma cell proliferation through Gi-coupled receptors: Role of ERK MAP kinase and phosphatidylinositol 3-kinase β. Cancer Lett. 2002. Jul 26;181(2):195-204. doi:10.1016/S0304-3835(02)00050-2
39. Young N, Van Brocklyn JR. Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness. Exp Cell Res. 2007. May 1;313(8):1615-27. doi:10.1016/j.yexcr.2007.02.009
40. Lepley D, Paik JH, Hla T, Ferrer F. The G protein-coupled receptor S1P2 regulates Rho/Rho kinase pathway to inhibit tumor cell migration. Cancer Res. 2005. May 1;65(9):3788-95. doi:10.1158/0008-5472.CAN-04-2311
41. Kapitonov D, Allegood JC, Mitchell C, et al. Targeting sphingosine kinase 1 inhibits Akt signaling, induces apoptosis, and suppresses growth of human glioblastoma cells and xenografts. Cancer Res. 2009. Sep 1;69(17):6915-23. doi:10.1158/0008-5472.CAN-09-0664
42. Rostami N, Nikkhoo A, Ajjoolabady A, et al. S1PR1 as a Novel Promising Therapeutic Target in Cancer Therapy. Mol Diagnosis Ther. 2019. Aug;23(4):467-487. doi:10.1007/s40291-019-00401-5
43. D’Aprile C, Prioni S, Mauri L, Prinetti A, Grassi S. Lipid rafts as platforms for sphingosine 1-phosphate metabolism and signalling. Cell Signal. 2021. Apr;80:109929. doi:10.1016/j.cellsig.2021.109929
44. Van Brocklyn JR, Young N, Roof R. Sphingosine-1-phosphate stimulates motility and invasiveness of human glioblastoma multiforme cells. Cancer Lett. 2003. Sep 10;199(1):53-60. doi:10.1016/S0304-3835(03)00334-3
45. Young N, Pearl DK, Van Brocklyn JR. Sphingosine-1-phosphate regulates glioblastoma cell invasiveness through the urokinase plasminogen activator system and CCN1/Cyr61. Mol Cancer Res. 2009. Jan;7(1):23-32. doi:10.1158/1541-7786.MCR-08-0061