Abstract 2897: Phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy
Authors: Gabriela Andrejeva, Gigin Lin, Harry G. Parkes, James Mui, Anne-Christine LF Wong Te Fong, Gema Vizcay-Barrena, Roland Fleck, Martin O. Leach, Yuen-Li Chung
Journal: Cancer Research (2015)

Abstract Autophagy is a cellular stress survival mechanism in which newly formed double-membrane vesicles target parts of the cytosol and organelles for lysosomal degradation to maintain cellular energy production. The source of the autophagosome membrane in starvation has been subject to much debate, but has not been investigated in the more clinically relevant setting of drug-induced autophagy where nutrients are available. By 1H-magnetic resonance spectroscopy (MRS) we show that autophagy, induced in human colorectal carcinoma cells HCT116 wt, HCT116 Bax-ko and HT29 by dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, by PI-103, a dual PI3K/mTOR inhibitor, or by starvation in medium lacking amino acids and growth factors, is associated with significant increases in membrane phospholipid phosphatidylcholine (PtdCho) (p≤0.02). 13-C labeled choline 13C-MRS revealed that these increases are due to de novo synthesis of PtdCho (p≤0.02) and are linked with increased expression of the rate-limiting enzyme, CDP:phosphocholine cytidylyltransferase (CCTα) in its active, membrane-bound form. Autophagic cells labeled with propargylcholine, a choline analog, had significant increase in propargylcholine phospholipid synthesis (p≤0.02) and vesicular structures consistent with autophagosomes when imaged by fluorescence confocal microscopy. Electron microscopy with immunogold labeling reveals the incorporation of newly synthesized propargylcholine phospholipids into the membranes of autophagic vacuoles and the digested material in autophagolysosomes, as well as endoplasmic reticulum, mitochondrial membranes and nuclear envelope as in control cells. To assess the role of PtdCho synthesis and CCTα in autophagy, we used Chinese hamster ovarian cells CHO-K1 MT58, with a temperature-sensitive mutation in the CCTα gene, resulting in reduced PtdCho synthesis at 40°C. PI-103 induced autophagy in wild-type CHO-K1 cells at both 33°C and 40°C. MT58 cells had normal autophagy induction with PI-103 at 33°C, but the autophagy marker LC3B-II was lower at 40°C than in the CHO-K1 cells. Loss of CCTα activity prevented PI-103-induced increase in PtdCho levels (p≤0.0003) and resulted in a reduced volume of autophagosomes in MT58 cells at 40°C (p≤0.01). Our evidence suggests that increased PtdCho synthesis in drug-induced autophagy provides membrane phospholipids for the growing autophagosomes and replaces phospholipids consumed from other organelles during autophagosome formation and degradation. Expression of CCTα, the rate-limiting enzyme of PtdCho synthesis, is increased in autophagy and its loss results in the inability of cells to maintain autophagosome formation. PtdCho synthesis might provide a new target for autophagy inhibition. Also, fluorescence imaging of propargylcholine labeling may offer a novel way to visualize newly formed autophagosome membranes in vitro. Citation Format: Gabriela Andrejeva, Gigin Lin, Harry G. Parkes, James Mui, Anne-Christine LF Wong Te Fong, Gema Vizcay-Barrena, Roland Fleck, Martin O. Leach, Yuen-Li Chung. Phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2897. doi:10.1158/1538-7445.AM2015-2897