Abstract 46: Interdependence of choline kinase and phospholipase D in human breast cancer cells

Abstract

Abstract One hallmark of cancer is an increase of cellular phosphocholine (PC) and total choline-containing compounds (tCho), which are closely related to malignant transformation, invasion and metastasis. Enzymes in choline metabolism present attractive targets that can be exploited for treatment. Choline kinase (Chk) is a cytosolic enzyme that catalyzes the phosphorylation of choline (Cho) to phosphocholine (PC) by using ATP in the presence of magnesium. Over-expression of Chk-α has been observed in breast, prostate and lung cancers, making it an obvious choice for targeting. Down-regulation of Chk-α results in a significant reduction of cell proliferation and increased differentiation in highly invasive MDA-MB-231 human breast cancer cells after siRNA-Chk transfection. Another potential target is phosphatidylcholine-specific phospholipase D (PC-PLD). Two mammalian isoforms of PLD, PLD1 and PLD2 have currently been identified. PLD1 is activated by G proteins such as ARF, Rho and Rac. Elevated PLD1 has been reported in gastric, renal, colon and breast carcinoma. We characterized the expression of PLD1 in metastatic MDA-MB-231 cells, poorly metastatic MCF-7 cells, and nonmalignant MCF-12A cells and used siRNA to transiently down-regulate PLD1 and Chk-α in MDA-MB-231 and MCF-7 cells. Forty-eight hours post-transfection cells were harvested for protein and RNA. To determine the effective knock-down of Chk-α or PLD1, about 30µg of protein was resolved on 7.5% acrylamide gel, transferred to nitrocellulose membrane and probed with antibody specific to either Chk-α or PLD1. The effects of Chk-α downregulation on PLD1 expression and the effects of PLD1 downregulation on Chk-α expression were determined by western blots, and the message was quantified with q-RT-PCR. Downregulation of Chk resulted in an increase of PLD1 in MDA-MB-231 cells stably expressing shRNA against Chk or transiently transfected with Chk siRNA. Similar data were obtained for MCF-7 cells. PLD1 levels were highest in the metastatic MDA-MB-231 cells. Effective downregulation of PLD1 was achieved with transient PLD1 siRNA transfection, which resulted in an increase of Chk-α in these cells. These data demonstrate the adaptability of cancer cells, the interdependence of Chk-α and PLD1, and the importance of multiple targeting to eliminate any adaptive compensatory effects that would allow cancer cells to survive. This work was supported by NIH P50 CA103175. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 46.