VERY LONG-CHAIN ACYL-CoA SYNTHETASE-3 (ACSVL3) PROMOTES THE MALIGNANT GROWTH BEHAVIOR OF U87 GLIOMA CELLS VIA CHANGES IN CELL CYCLE WITHOUT AFFECTING APOPTOSIS

Abstract

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. Gamma-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.

Depleting glioblastoma cells of very long-chain acyl-CoA synthetase 3 (ACSVL3) produces metabolic alterations in non-lipid pathways

“…In particular, ACSVL3 overexpression resulted in increased sphingolipid synthesis. We hypothesized that this contributes to the rapid growth phenotype of these glioblastoma cells (8). Depletion of ACSVL3 in U87 cells restores them to a more normal, less malignant growth phenotype, suggesting that targeting of this enzyme may have therapeutic benefits.…”

“…We previously reported that while membrane phospholipid synthesis was not significantly different in U87-KO cells vs. U87MG cells, there were significant alterations in sphingolipid metabolism when ACSVL3 levels were depleted (7). We also reported that malignant U87MG cells were quite resistant to apoptosis, and that increased apoptosis in U87-KO cells could not explain the slower growth rate of these cells (8). In contrast, the cell cycle was dramatically altered in ACSVL3-deficient U87-KO cells (8), a phenomenon that may be related to the alterations in sphingolipid metabolism (7).…”

“…We also reported that malignant U87MG cells were quite resistant to apoptosis, and that increased apoptosis in U87-KO cells could not explain the slower growth rate of these cells (8). In contrast, the cell cycle was dramatically altered in ACSVL3-deficient U87-KO cells (8), a phenomenon that may be related to the alterations in sphingolipid metabolism (7). In this report, we show that KO of ACSVL3 in U87MG cells produces significant changes in non-lipid pathways of intermediary metabolism as well.…”