Abstract 1255: Role of choline in adaptive mechanisms in choline phospholipid metabolism of human breast cancer cells

Abstract

Abstract Proton magnetic resonance spectroscopy studies of cancer have consistently detected increased cellular phosphocholine (PC) and total choline-containing compounds (tCho), which are closely related to malignant transformation, invasion and metastasis [1]. Enzymes in choline metabolism present attractive targets that can be exploited for treatment [2]. Two of these enzymes, choline kinase-α (Chk) and phosphatidylcholine specific phospholipase D (PC-PLD) have been associated with increased malignancy [1, 3]. Chk is a cytosolic enzyme that is responsible for phosphorylation and converting free choline to phosphocholine. Two mammalian isoforms of PC-PLD, PLD1 and PLD2 have currently been identified. While PLD2 is restricted to cytoplasm, PLD1 shuttles between cytoplasm and membrane upon activation by G proteins such as ARF, Rho and Rac [4]. Here we used small interfering RNA (siRNA) against Chk and PLD1 alone or in combination in triple negative MDA-MB-231 human breast cancer cells to silence these two genes. Effective downregulation was confirmed at both protein and mRNA levels. However, downregulation of Chk with siRNA resulted in increased PLD1 expression, and downregulation of PLD1 resulted in increased Chk expression, typifying the ability of cancer cells to adapt. We sought to further understand mechanisms underlying this interdependence by changing the concentrations of free choline in the media. We observed a reduction in the increase of PLD1 protein expression and mRNA levels following Chk downregulation with higher concentration of free choline (107.1µM), suggesting that free choline plays a role in mediating the increase of PLD1 observed following Chk downregulation. Understanding the complex networks of pathways that participate following downregulation of a specific target is critical in a complex disease like cancer. These participating pathways provide novel opportunities to target in concert with the primary target to achieve improved control and minimize compensatory responses that allow cancer cells to survive or adapt. This is especially important if the downregulation of one ‘oncogenic target’, e.g. Chk, results in the upregulation of another, e.g. PLD1. Acknowledgements: This work was supported by NIH P50 CA103175.