Abstract B06: TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism
Authors: Priolo Carmen , Ricoult Stéphane J. H. , Khabibullin Damir , Filippakis Harilaos , Yu Jane , Manning Brendan , Clish Clary , Henske Elizabeth P. 
Published: May 2016

Abstract
Tuberous Sclerosis Complex (TSC) is an autosomal dominant disease characterized by multi-organ proliferation of TSC2-deficient cells with aberrant activation of the mechanistic/mammalian Target of Rapamycin Complex 1 (mTORC1), a master regulator of cell growth and metabolism. Up to 80% of women with TSC develop lymphangioleiomyomatosis (LAM), which consists of diffuse proliferation of TSC2-deficient smooth muscle-like cells with progressive cystic destruction of the lung. LAM can also occur as a sporadic disorder of women. A downstream effector of mTORC1, the Sterol Regulatory Element-Binding Protein (SREBP), regulates the transcription of de novo fatty acid synthesis enzymes in TSC2-deficient cells. The impact of loss of the TSC proteins on the lipidome and how complex lipid species are affected by rapamycin and its analogs are unknown. Here, we report the first systematic study of the TSC lipidome, revealing unexpected findings.
Using mass spectrometry, we profiled 131 complex lipid species discovered elevated levels (p&lt;0.05) of 4 lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, C20:4) in the plasma of LAM patients compared with healthy control women. LPC are a class of bioactive lipids generated by phospholipase A (PLA) activity. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro pre-clinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid, including the LPC precursors phosphatidylcholines (PC) and sphingolipids, and neutral lipid species, including triacylglycerols and cholesterol esters.
To determine whether TSC2-deficient cells generate LPC using de novo synthesized PC, TSC2-deficient and TSC2-expressing cells were labeled with deuterated choline (choline chloride 1,1,2,2-d4) for 6 hours, and lipids isolated to measure choline incorporation into two saturated LPC (C16:0 and C18:0) and two parent PC (C32:0 and C34:0) species. Incorporation of deuterated choline into PC and LPC was greater than 2 fold higher in TSC2-deficient cells compared to TSC2-expressing controls (p&lt;0.05).
Surprisingly, treatment with rapamycin, the mTOR catalytic inhibitor Torin1, or downregulation of the mTORC1-regulated lipogenic transcription factor SREBP (Sterol Regulatory Element-Binding Protein) did not suppress LPC in TSC2-deficient cells, while they suppressed other phospholipid species. Finally, pharmacologic and genetic approaches revealed that inhibition of distinct isoforms of PLA2 may decrease the proliferation of TSC2-deficient but not TSC2-expressing cells.
Collectively, these results demonstrate for the first time that TSC2-deficient cells have enhanced choline phospholipid metabolism, revealing a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies.
Citation Format: Carmen Priolo, Stéphane J. H. Ricoult, Damir Khabibullin, Harilaos Filippakis, Jane Yu, Brendan Manning, Clary Clish, Elizabeth P. Henske. TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B06.