PHOSPHATIDYLCHOLINE BIOSYNTHESIS IS INVOLVED IN AUTOPHAGY REGULATION IN RAS-TRANSFORMED INTESTINAL EPITHELIAL CELLS

Abstract

Ras oncogene increased CTP-phosphocholine cytidyltransferase α (CCTα) expression and phosphatidylcholine (PC) biosynthesis that were required for cell proliferation and survival. Ras-transformed intestinal epithelial cells (IEC-ras) have increased autophagy, which could be related to increased PC because phagophore membrane synthesis and PC synthesis take place at the endoplasmic reticulum (ER). Thus, we examined whether CCTα and PC synthesis are required for autophagy. RNAi silencing of CCTα in IEC-ras caused increased expression of p62 and LC3-II, indicative of impairment of autophagy. Decreased PC biosynthesis by choline depletion of IEC-ras also caused accumulation of p62/SQSTM1 and LC3-II, and inhibited cell growth. Choline depletion of non-malignant IEC cells had no effect on these parameters. Chloroquine did not alter p62/SQSTM1 or LC3-II in IEC-ras cultured in the absence of choline, indicating that autophagy is blocked before autophagosome-lysosome fusion. Autophagy was restored in choline-depleted IEC-ras34 by addition of lysophosphatidylcholine, which can be converted to PC. These data show that inhibition of PC synthesis is required to sustain autophagy and proliferation of IEC-ras.