Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras V12 in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feed-forward amplification of cancer survival signaling pathways.
|Number of pages||11|
|State||Published - Jan 2010|
Bibliographical noteFunding Information:
Lipids were extracted in chloroform/methanol, dried under a stream of N2 gas, and analysed using electrospray ionization-tandem mass spectrometry at Kansas Lipidomics Research Center (supported by National Science Foundation (EPS 0236913, MCB 0455318, DBI 0521587), Kansas Technology Enterprise Corporation, K-IDeA Networks of Biomedical Research Excellence (INBRE) of National Institute of Health (P20RR16475), Kansas State University and the Functional Genomics Consortium initiative of Kansas State University’s Targeted Excellence Program). Lipid species were detected using the following scans: PC and lysoPC, [M + H]+ ions in positive ion mode with Precursor of 184.1 (Pre 184.1); PE and lysoPE, [M + H]+ ions in positive ion mode with neutral loss of 141.0 (NL 141.0); PG, [M + NH4]+ in positive ion mode with NL 189.0 for PG; lysoPG, [M−H]− in negative mode with Pre 152.9; PI, [M + NH4]+ in positive ion mode with NL 277.0; PS, [M + NH4]+ in positive ion mode with NL 185.0; and PA, [M + NH4]+ in positive ion mode with NL 115.0. The scan speed was 50 or 100 u/s. The collision gas pressure was set at 2 (arbitrary units). The collision energies, with nitrogen in the collision cell, were + 28 V for PE, + 40 V for PC, + 25 V for PI, PS and PA and + 20 V for PG. Declustering potentials were + 100 V for PE, PC, PA, PG, PI and PS. Entrance potentials were + 15 V for PE, + 14 V for PC, PI, PA, PG and PS. Exit potentials were + 11 V for PE, + 14 V for PC, PI, PA, PG and PS. The mass analysers were adjusted to a resolution of 0.7 u full width at half height. For each spectrum, 9–150 continuum scans were averaged in multiple channel analyser mode. The source temperature (heated nebulizer) was 100 1C, the interface heater was on, + 5.5 kV or −4.5 kV were applied to the electrospray capillary, the curtain gas was set at 20 (arbitrary units) and the two ion source gases were set at 45 (arbitrary units). The background of each spectrum was subtracted, the data were smoothed and peak areas were integrated using a custom script and Applied Biosystems Analyst software (Foster City, CA, USA). The lipids in each class were quantified in comparison with the two internal standards of that class. The first and typically every eleventh set of mass spectra were acquired on the internal standard mixture only. Peaks corresponding to the target lipids in these spectra were identified and molar amounts calculated in comparison with the internal standards on the same lipid class. To correct for chemical or instrumental noise in the samples, the molar amount of each lipid metabolite detected in the ‘internal standards only’ spectra was subtracted from the molar amount of each metabolite calculated in each set of sample spectra. The data from each ‘internal standards only’ set of spectra were used to correct the data from the following 10 samples. Finally, the data were corrected for the fraction of the sample analysed and normalized to the sample ‘dry weights’.
We gratefully acknowledge helpful discussions with Drs Binks Wattenberg, Mary Roth and Otto Grubraw. This work was supported by the James Graham Brown Cancer Center and by the following grants: NIH 1 R01 CA11642801 (JC) and the Kentucky Lung Cancer Research Program (JC).
- Phosphatidic acid
ASJC Scopus subject areas
- Molecular Biology
- Cancer Research