Identification of magnetic resonance detectable metabolic changes associated with inhibition of phosphoinositide 3-kinase signaling in human breast cancer cells Mounia Beloueche-Babari, 1 L. Elizabeth Jackson, 1 Nada M.S. Al-Saffar, 1 Suzanne A. Eccles, 2 Florence I. Raynaud, 2 Paul Workman, 2 Martin O. Leach, 1 and Sabrina M. Ronen 1 1 Cancer Research UK Clinical Magnetic Resonance Research Group, The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust; 2 Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom Abstract Phosphoinositide 3-kinase (PI3K) is an attractive target for novel mechanism-based anticancer treatment. We used magnetic resonance (MR) spectroscopy (MRS) to detect biomarkers of PI3K signaling inhibition in human breast cancer cells. MDA-MB-231, MCF-7, and Hs578T cells were treated with the prototype PI3K inhibitor LY294002, and the 31 P MR spectra of cell extracts were monitored. In every case, LY294002 treatment was associated with a significant decrease in phosphocholine levels by up to 2- fold (P < 0.05). In addition, a significant increase in glycerophosphocholine levels by up to 5-fold was also observed (P V 0.05), whereas the content of glycerophos- phoethanolamine, when detectable, did not change sig- nificantly. Nucleotide triphosphate levels did not change significantly in MCF-7 and MDA-MB-231 cells but de- creased by f1.3-fold in Hs578T cells (P = 0.01). The changes in phosphocholine and glycerophosphocholine levels seen in cell extracts were also detectable in the 31 P MR spectra of intact MDA-MB-231 cells following exposure to LY294002. When treated with another PI3K inhibitor, wortmannin, MDA-MB-231 cells also showed a significant decrease in phosphocholine content by f1.25- fold relative to the control (P < 0.05), whereas the levels of the remaining metabolites did not change significantly. Our results indicate that PI3K inhibition in human breast cancer cells by LY294002 and wortmannin is associated with a decrease in phosphocholine levels. [Mol Cancer Ther 2006;5(1):187 – 96] Introduction The phosphoinositide 3-kinase (PI3K) pathway is a signaling cascade that is activated following association of PI3K with activated Ras proteins, stimulated growth factor receptors, and G protein – coupled receptors as well as via chemokine receptors and adhesion molecules (1, 2). PI3Ks are a family of lipid kinases that catalyze the formation of phosphatidylinositol 3,4-bisphosphate and phosphatidyl- inositol 3,4,5-triphosphate; these products selectively bind to many signaling proteins, including phosphoinositide- dependent kinase 1, protein kinase B/Akt, and Rac, leading to activation of downstream signaling pathways (1, 2). The PI3K family comprises three main classes: I, II, and III (3, 4); its members mediate many key cellular processes, includ- ing growth and survival, cell cycle entry, adhesion, and migration (5). Growing evidence suggests that aberrant PI3K signaling could play a crucial role in the development of many human cancers (3–7). Ras proteins are activated in f30% of all human cancers (8). Moreover, activation of Akt has been reported in a range of human tumors (7, 9). Amplification and mutation of the PI3KCA gene, which encodes the PI3K catalytic subunit p110a, has been identified in several types of cancer, including ovarian, cervical, breast, and brain tumors (10–13). Furthermore, deletion or reduced expres- sion of the tumor suppressor PTEN, which encodes a lipid phosphatase that reverses PI3K activity, is very common and leads to sustained activation of PI3K signaling (3, 4, 7, 14). Based on these observations, it is now believed that PI3K signaling is an attractive target for mechanism-based anticancer treatment (15, 16). Detecting biomarkers of target inhibition is critical for assessing the efficacy of treatment and for correlating antitumor effects with target suppression and is playing an increasingly important part in the clinical evaluation of novel molecular therapeutics (17, 18). Current techni- ques for measuring proof-of-concept, pharmacodynamic, or response biomarkers are surgically invasive. Thus, noninvasive end points would be extremely valuable (17). Received 9/22/05; revised 10/17/05; accepted 11/4/05. Grant support: Cancer Research UK grants C1060/A808 (M. Beloueche- Babari, L.E. Jackson, M.O. Leach, and S.M. Ronen) and C309/A2187 (P. Workman, F.I. Raynaud, and S.A. Eccles) and Association for International Cancer Research grant 03-304 (N.M.S. Al-Saffar). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: P. Workman is a Cancer Research UK Life Fellow. S.M. Ronen is currently at the Department of Experimental Diagnostic Imaging, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030-4095. Requests for reprints: Mounia Beloueche-Babari, Cancer Research UK Clinical Magnetic Resonance Research Group, Institute of Cancer Research and Royal Marsden NHS Foundation Trust, Downs Road, Sutton, Surrey SM2 5PT, United Kingdom. Phone: 44-208-661-3728; Fax: 44-208-661-0846. E-mail: Mounia.Beloueche-Babari@icr.ac.uk Copyright C 2006 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-03-0220 187 Mol Cancer Ther 2006;5(1). January 2006 Research. on December 22, 2017. © 2006 American Association for Cancer mct.aacrjournals.org Downloaded from