Abstract 1195: Ethanolamine kinase-1 and phosphoethanolamine are potential diagnostic markers and therapeutic targets in breast cancer
Authors: Shah Tariq , Krishnamachary Balaji , Wildes Flonne , Wijnen Jannie , Glunde Kristine , Bhujwalla Zaver M. 
Published: August 2015

Abstract
Increased phosphoethanolamine (PE) has been observed in tumors almost as consistently as increased phosphocholine (PC), but the role of PE in cancer is relatively unexplored. Our ongoing studies have shown that choline kinase (ChK)-α, the enzyme that forms PC from choline, has a dual choline/ethanolamine kinase activity but ChK-β has no significant role in maintaining PE levels in vivo. We observed that ethanolamine kinase (EtnK)-1siRNA significantly reduces PE levels in triple negative MDA-MB-231 human breast cancer cells. We further investigated the role of ethanolamine kinase-2 (EtnK-2) in contributing to the increased PE observed in cancers, and the effect of various small interfering RNA (siRNA) combinations downregulating ChK and EtnK on cell viability, as potential therapeutic strategies in these triple negative breast cancer cells.
Cells were cultured in RPMI-1640 medium supplemented with 20μM choline and 50μM ethanolamine. While 50nM siRNA was used in all individual siRNA treatment, for combination siRNA treatment 50nM of each specific siRNA was used. Silencing of 80-90% message was confirmed using qRT-PCR for the siRNAs used. Approximately 40 million cells were harvested 48h post transfection and cell extracts were prepared using a dual-phase extraction method based on methanol/chloroform/water (1/1/1; v/v/v). 31P MR spectra were acquired on water soluble fraction and metabolites were quantitated. For assaying cell viability, 4000 cells per well were plated in a 96 well plate and transfected with 50nM siRNA and viability assessed 96h post transfection.
PC decreased consistently when cells were treated with Chk-α siRNA or in the presence of high levels of ethanolamine in the medium showing dual choline/ethanolamine kinase activity of ChK-α. In cells treated with EtnK-1 siRNA, a significant reduction in PE levels was observed. ChK-β or EtnK-2 siRNA treatment did not affect PC or PE levels. These results indicate that ChK-β and EtnK-2 do not have significant choline or ethanolamine kinase activity in these breast cancer cells. Etnk-1 is the major contributor to PE levels in these breast cancer cells with some contribution from Chk-α. ChK-β siRNA did not have a significant effect on cell viability whereas ChK-α and EtnK1 siRNA resulted in ∼ 60% and 50% reduction of cell viability respectively. Combined treatment with ChK-α+ EtnK-1 siRNA was not more effective than treatment with the individual siRNA alone, suggesting that both siRNA act through the same path in decreasing cell viability. The reduction of cell viability caused by treatment with EtnK-1 siRNA and its major contribution to elevated PE levels in tumors indicates its therapeutic potential that warrants further investigation. Additionally, like radiolabeled choline, radiolabeled ethanolamine may be useful in detecting breast and other cancers with PET imaging.
Acknowledgements: Supported by P50 CA103175.
Citation Format: Tariq Shah, Balaji Krishnamachary, Flonne Wildes, Jannie Wijnen, Kristine Glunde, Zaver M. Bhujwalla. Ethanolamine kinase-1 and phosphoethanolamine are potential diagnostic markers and therapeutic targets in breast cancer. [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 1195. doi:10.1158/1538-7445.AM2015-1195