The Watkins Lab has observed that when U87 human malignant glioma cells are deficient for a particular acyl- CoA synthetase, ACSVL3, the cells had significantly lower cholesterol levels than did wild-type U87 cells. Depleting U87 cells of ACSVL3 decreased their malignant growth properties in vitro and decreased their tumorigencity in mice. Because there is no known role for any acyl-CoA synthetase in cholesterol metabolism, exactly how ACSVL3 affects cholesterol homeostasis and how this in turn affects malignancy is unknown. I hypothesize that depletion of ACSVL3 affects the sterol response element binding proteins (SREBPs) that control the transcription of genes important in cholesterol homeostasis and fatty acid synthesis, and the lack of cholesterol disrupts lipid raft formation and cellular signaling from these rafts. To answer this, I propose to 1) determine the effect of ACSVL3 depletion on cholesterol levels in human glioma cells, 2) determine how ACSVL3 regulates cholesterol homeostasis in cancer cells, and 3) determine how ACSVL3 depletion affects lipid raft formation and signaling. I plan on accomplishing the first aim by measuring intracellular cholesterol levels, and see how the growth and the tumorigenic properties are affected by repleting or depleting the cells of cholesterol. In the second aim, I use qRT-PCR to observe changes in transcription levels of SREBP target genes in the control U87 cells and compare them to ACSVL-deficient cells. I will also assess translocation of SREBPs to the nucleus and the potential involvement of SCAP and Insig proteins. Aim 3 will be accomplished by using fluorescent cholesterol markers to observe the changes in lipid raft morphology and Western Blotting and immunofluorescence will allow me to look at differences in c-Met levels and raft marker proteins. Reporter constructs will be used in live cell imaging studies to assess effects on signaling via c-Met, PI3 kinase, and Akt. This research may lead to new therapeutic strategies for human cancers and further our understanding of the role of lipid metabolism in malignancy.

In both children and adults, malignant gliomas account for about half of all brain tumors, and are associated with a poor prognosis despite aggressive therapy; thus, new therapeutic measures are needed. In this proposal I will investigate why a reduction in cellular cholesterol levels, brought about by depleting cells of the fatty acid metabolism enzyme ACSVL3, decreases the malignant behavior of human glioma cells. Results of these studies may suggest new pathways to target for pharmacologic treatment of gliomas and perhaps other cancers.