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Selective Inhibition of Choline Kinase Simultaneously Attenuates MAPK and PI3K/AKT Signaling

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Selective Inhibition of Choline Kinase Simultaneously Attenuates MAPK and PI3K/AKT Signaling Oncogene (2010) 29, 139–149 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling A Yalcin1,2, B Clem1, S Makoni1, A Clem1, K Nelson1, J Thornburg1, D Siow1, AN Lane1, SE Brock1, U Goswami1, JW Eaton1, S Telang1 and J Chesney1 1Department of Biochemistry and Molecular Biology, James Graham Brown Cancer Center (Molecular Targets Program), University of Louisville, Louisville, KY, USA and 2Department of Biochemistry, School of Veterinary Medicine, Uludag University, Bursa, Turkey Choline is an essential anabolic substrate for the synthesis Introduction of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production Human cancers have been found to transport and of phosphatidylcholine, the major phospholipid constituent of phosphorylate increased choline relative to adjacent membranes and substrate for the synthesis of lipid signaling normal tissues (Degani et al., 1986; Onodera et al., 1986; molecules. Nuclear magnetic resonance (NMR)-based meta- Ackerstaff et al., 2003; Eliyahu et al., 2007; Glunde and bolomic studies of human tumors have identified a marked Bhujwalla, 2007). The high concentration of phospho- increase in the intracellular concentration of phosphocholine choline observed in cancers is due, in part, to the growth relative to normal tissues. We postulated that the observed factor-activated Ras and phosphatidylinositol 3-kinase intracellular pooling of phosphocholine may be required to (PI3K) signaling cascades that stimulate the initial sustain the production of the pleiotropic lipid second enzyme of the cytidylyl diphosphate-choline (or Kenne- messenger, phosphatidic acid. Phosphatidic acid is generated dy) pathway, choline kinase, through the Rho GTPases from the cleavage of phosphatidylcholine by phospholipase D2 (Ramirez de Molina et al., 2002a, 2004, 2005). Choline and is a key activator of the mitogen-activated protein kinase kinase (ATP/choline phosphotransferase) catalyses the (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT phosphorylation of choline by adenosine triphosphate in survival signaling pathways. In this study we show that the the presence of Mg2 þ and has been found to be activated steady-state concentration of phosphocholine is increased by in malignant cells and tumors of the lung, colon, breast, V12 the ectopic expression of oncogenic H-Ras in immortalized prostate, cervix and ovaries (Ramirez de Molina et al., human bronchial epithelial cells. We then find that small 2002b; Iorio et al., 2005). In mammalian cells, there exist interfering RNA (siRNA) silencing of choline kinase expres- at least three isoforms of choline kinase—the mRNA sion in transformed HeLa cells completely abrogates the high splice variants choline kinase-a1 and choline kinase-a2, concentration of phosphocholine, which in turn decreases and choline kinase-b, which is encoded by a separate phosphatidylcholine, phosphatidic acid and signaling through gene (Aoyama et al., 2002, 2004). Inhibition of choline the MAPK and PI3K/AKT pathways. This simultaneous kinase-a and choline kinase-b mRNA expression in reduction in survival signaling markedly decreases the breast adenocarcinoma cells using choline kinase small anchorage-independent survival of HeLa cells in soft agar interfering RNA (siRNA) reduces intracellular phos- and in athymic mice. Last, we confirm the relative importance phocholine, which in turn decreases cellular prolifera- of phosphatidic acid for this pro-survival effect as phospha- tion and promotes differentiation (Glunde et al., 2005). tidic acid supplementation fully restores MAPK signaling and Furthermore, transfection with choline kinase small partially rescues HeLa cells from choline kinase inhibition. hairpin RNA plasmids specific for choline kinase-a Taken together, these data indicate that the pooling of selectively reduces proliferation and increases apoptosis phosphocholine in cancer cells may be required to provide a in breast adenocarcinoma cells, but not in normal ready supply of phosphatidic acid necessary for the feed- human mammary epithelial cells (Banez-Coronel et al., forward amplification of cancer survival signaling pathways. 2008). Last, a global analysis of the tumors of patients Oncogene (2010) 29, 139–149; doi:10.1038/onc.2009.317; with early-stage non-small cell lung cancer recently published online 26 October 2009 established an association between choline kinase-a Keywords: ras; phosphatidic acid; phosphocholine mRNA overexpression and poor clinical outcome (Ramirez de Molina et al., 2007). On the basis of these observations, choline kinase and, in particular, choline kinase-a has been proposed as a prognostic marker for Correspondence: Dr J Chesney, Department of Biochemistry and cancer progression as well as a molecular target for the Molecular Biology, University of Louisville, 580 South Preston Street, development of novel cancer chemotherapeutic agents 204E, Louisville, KY 40202, USA. et al. et al. E-mail: [email protected] (Ackerstaff , 2003; Glunde , 2006; Malito Received 21 April 2009; revised 21 August 2009; accepted 24 August et al., 2006; Eliyahu et al., 2007; Glunde and Bhujwalla, 2009; published online 26 October 2009 2007; Ramirez de Molina et al., 2007). CK inhibition on MAPK and PI3K/AKT signaling A Yalcin et al 140 The precise rationale for the high intracellular then transformed using the telomerase catalytic subunit phosphocholine levels observed in cancer is not well (hT), simian virus 40 large T antigen (LT) and an understood. Choline kinase commits choline to the oncogenic allele of ras (H-RasV12) (Soejima et al., 2003). Kennedy pathway for the biosynthesis of phosphatidyl- In previous studies, we have confirmed the protein choline, and neoplastic cells have a great need for such expression of the hT, simian virus 40 LT and H-RasV12 membrane phospholipids as a result of increased using western blot analysis (Telang et al., 2006, 2007). membrane turnover, endosome formation required for The hT/LT cells are immortalized as they have under- growth factor signaling (Vieira et al., 1996; Li et al., gone >100 doublings and the hT/LT/Ras cells are 1997) and the secretion of immune-modulating exo- transformed as they have the capacity for anchorage- somes (Iero et al., 2008). However, phosphatidylcholine independent growth in soft agar and athymic mice produced from phosphocholine is also a substrate for (Soejima et al., 2003). Importantly, the hT/LT and hT/ the production of several lipid second messengers, LT/Ras cells proliferate at a similar rate, which is including lyso-phosphatidylcholine, diacylglycerol, approximately twice that of primary normal human lyso-phosphatidic acid and phosphatidic acid (Exton, bronchial epithelial cells (with epidermal growth factor 1990). Phosphatidic acid, generated from the cleavage of stimulation) (Telang et al., 2006, 2007). We prepared phosphatidylcholine by phospholipase D2 (Buchanan trichloroacetic acid extracts of the primary, hT/LT- et al., 2005), has emerged as an essential upstream and immortalized and ras-transformed bronchial epithelial downstream activator of the Ras signaling pathway (Rizzo cells in exponential growth phase and recorded nuclear and Romero, 2002; Foster and Xu, 2003; Hancock, 2007). magnetic resonance (NMR) spectra at 14.1 T on a For example, transfection of Ewing’s sarcoma cells with Varian Inova NMR spectrometer (Varian, Palo Alto, phospholipase D2 siRNA markedly reduces activating CA, USA) at 20 1C using a 901 excitation pulse, an phosphorylations of extracellular signal-regulated kinase acquisition time of 2 s and a relaxation delay of 3 s. (ERK) and AKT, the two signaling proteins required for Under these conditions, the protons are essentially full survival (Nozawa et al., 2005). Phosphatidic acid is a relaxed, as shown by independent measurements of T1. negatively charged phospholipid that can function as a We observed a marked increase in the phosphocholine lipid anchor by directly binding to positively charged after immortalization that further increased with trans- portions of effector proteins (Zhao et al., 2007). Phospha- formation (Figure 1, normal human bronchial epithelial, tidic acid activates and amplifies Ras signaling by: (1) 33±7; hT/LT, 91±11; hT/LT/Ras, 179±21 nmol/ recruiting the guanine nucleotide-exchange factor, son of 5 Â 106 cells). These data show that the sequential sevenless (Sos) (Zhao et al., 2007), and the serine/threonine immortalization and transformation of human bron- kinase, Raf-1 (Rizzo et al., 1999, 2000), to the plasma chial epithelial cells is mirrored by a step-wise increase in membrane; (2) stimulating endosome formation necessary the steady-state concentration of phosphocholine. for MAP kinase activation (Rizzo and Romero, 2002); and (3) activating the mammalian target of rapamycin complex (mTOR) kinase (Fang et al., 2001; Toschi et al., 2009). Choline kinase-a is required for the metabolic pooling We postulated that phosphocholine may serve as an of phosphocholine in HeLa cells essential metabolic hub not only for membrane phos- We next transfected the hT/LT/Ras cells with several pholipid synthesis, but also for the amplification of choline kinase-a siRNA species and measured choline neoplastic signaling cascades required for survival and/ kinase mRNA expression by real-time reverse transcrip- or growth. In the following study, we will provide
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