17-allyamino-17-demethoxygeldanamycin treatment results in a magnetic resonance spectroscopy-detectable elevation in choline-containing metabolites associated with increased expression of choline transporter SLC44A1 and phospholipase A2

Abstract

Introduction: 17-allyamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is currently in clinical trials in breast cancer. However, 17-AAG treatment often results in inhibition of tumor growth rather than shrinkage, making detection of response a challenge. Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) are noninvasive imaging methods than can be used to monitor metabolic biomarkers of drug-target modulation. This study set out to examine the MRS-detectable metabolic consequences of Hsp90 inhibition in a breast cancer model.

Methods: MCF-7 breast cancer cells were investigated, and MRS studies were performed both on live cells and on cell extracts. (31)P and (1)H MRS were used to determine total cellular metabolite concentrations and (13)C MRS was used to probe the metabolism of [1,2-(13)C]-choline. To explain the MRS metabolic findings, microarray and RT-PCR were used to analyze gene expression, and in vitro activity assays were performed to determine changes in enzymatic activity following 17-AAG treatment.

Results: Treatment of MCF-7 cells with 17-AAG for 48 hours caused a significant increase in intracellular levels of choline (to 266 ± 18% of control, P = 0.05) and phosphocholine (PC; to 181 ± 10% of control, P = 0.001) associated with an increase in expression of choline transporter SLC44A1 and an elevation in the de novo synthesis of PC. We also detected an increase in intracellular levels of glycerophosphocholine (GPC; to 176 ± 38% of control, P = 0.03) associated with an increase in PLA2 expression and activity.

Conclusions: This study determined that in the MCF-7 breast cancer model inhibition of Hsp90 by 17-AAG results in a significant MRS-detectable increase in choline, PC and GPC, which is likely due to an increase in choline transport into the cell and phospholipase activation. (1)H MRSI can be used in the clinical setting to detect levels of total choline-containing metabolite (t-Cho, composed of intracellular choline, PC and GPC). As Hsp90 inhibitors enter routine clinical use, t-Cho could thus provide an easily detectable, noninvasive metabolic biomarker of Hsp90 inhibition in breast cancer patients.

Figure 1

Schematic of choline phospholipid metabolism. Schematic drawing of choline phospholipid metabolism and its regulatory enzymes illustrates the metabolic reactions associated with modulation of choline-containing metabolites. CCT, CTP:phosphocholine cytidylyltransferase; CDP-choline, cytidine diphosphate-choline; ChoK, choline kinase; CPT, diacylglycerol cholinephosphotransferase; GDPD, glycerophosphocholine phosphodiesterase; GPC, glycerophosphocholine; LPL, lysophospholipase; PC, phosphocholine; PLA, phospholipase A; PLC, phospholipase C; PLD, phospholipase D; PtdCho, phosphatidylcholine.

Figure 2

Effect of 17-AAG on cell proliferation and Hsp90 client protein levels. (a) Western blot analysis showing depletion of Hsp90 client proteins total-Akt and c-Raf following 17-AAG treatment of MCF-7 cells for 48 hours. Actin was used as a loading control. (b) Results of WST-1 assay showing 17-AAG effects on MCF-7 cell proliferation over a 48-hour treatment period. 17-AAG, 17-allyamino-17-demethoxygeldanamycin; Hsp90, heat shock protein 90.

Figure 3

Magnetic resonance spectroscopy detection of increased total and de novo PC in live, perfused cells following 17-AAG treatment. (a) ³¹P spectra of live, perfused MCF-7 cells illustrating an increase in PC after 48 hours of 17-AAG treatment. (b) ¹³C spectral array depicting the build-up of [1,2-¹³C]-PC in perfused MCF-7 cells over a 14-hour period. (c) Graph of build-up of de novo [1,2-¹³C]-PC in control (blue) and 17-AAG-treated (red) MCF-7 cells over 14 hours of exposure to [1,2-¹³C]-choline. The data represent an average of three repeats. 17-AAG, 17-allyamino-17-demethoxygeldanamycin; GPC, glycerophosphocholine; NTP, nucleoside triphosphate; PC, phosphocholine; Pi, inorganic phosphate; PPM, parts per million.

Figure 4

Representative ³¹P, ¹³C, and ¹H spectra of cell extracts. Representative spectra of control (bottom) and 17-AAG-treated (top) MCF-7 cells illustrate increased PC, intracellular choline, and GPC levels and unchanged PtdCho levels. (a) ¹³P spectra of the aqueous cell extract fraction depicting an increase in total PC and GPC levels after treatment. (b) ¹³C spectra of the aqueous cell extract fraction depicting an increase in de novo synthesized PC, GPC, and intracellular choline. (c) ³¹P spectra of the lipid cell extract fraction depicting constant PtdCho with 17-AAG treatment. (d) ¹H spectra of the aqueous cell extract fraction depicting an increase in the intracellular choline, PC, and GPC concentrations. 17-AAG, 17-allyamino-17-demethoxygeldanamycin; GPC, glycerophosphocholine; NTP, nucleoside triphosphate; PC, phosphocholine; PCr, phosphocreatine; Pi, inorganic phosphate; PPM, parts per million; PtdCho, phosphatidylcholine; PtdEth, phosphatidylethanol.