Oleate-mediated activation of phospholipase D and mammalian target of rapamycin (mTOR) regulates proliferation and rapamycin sensitivity of hepatocarcinoma cells

A high-fat diet and obesity are associated with increased risk of liver cancer. Because increased delivery of NEFA to the liver occurs in these conditions, we investigated the involvement of the unsaturated fatty acid oleate in hepatocarcinoma cell proliferation using human-derived hepatocarcinoma cell lines as model systems. Western blotting, FACS analysis and [H-3]thymidine incorporation were used to study the signalling pathways and the proliferation of cells cultured for up to 72 h with or without a concentration of oleate considered to be relevant to human pathophysiology (50 mu mol/l) or a concentration considered elevated (1 mmol/l). In HepG2 cells, proliferation was increased in the presence of 50 mu mol/l oleate, but was decreased at 1 mmol/l. This differential effect was correlated with the activation of the mammalian target of rapamycin complex 1 (mTORC1) and with increased translation of cell cycle regulators. Oleate-mediated mTORC1 activation required phospholipase D activation, which produces phosphatidic acid and is known to render mTORC1 rapamycin resistant. Remarkably, rapamycin resistance was found to affect specifically the mTORC1/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) branch of the mTORC1 pathway in the presence of 50 mu mol/l oleate. Furthermore, inhibition of phosphatidic acid production abolished oleate-induced increases in mTORC1 activity and cyclin A production. Importantly, the same differential effects of oleate on mTORC1 activation, cell cycle regulators and rapamycin resistance were found in SK-Hep1 cells. Oleate stimulates mTORC1 activation and rapamycin resistance. We propose that rapamycin-derived mTOR inhibitors are likely to be of limited therapeutic use to restrain hepatic tumour growth, particularly in the context of associated obesity.