Noninvasive detection of target modulation following phosphatidylinositol 3-kinase inhibition using hyperpolarized 13C magnetic resonance spectroscopy

Abstract

Numerous mechanism-based anticancer drugs that target the phosphatidylinositol 3-kinase (PI3K) pathway are in clinical trials. However, it remains challenging to assess responses by traditional imaging methods. Here, we show for the first time the efficacy of hyperpolarized (13)C magnetic resonance spectroscopy (MRS) in detecting the effect of PI3K inhibition by monitoring hyperpolarized [1-(13)C]lactate levels produced from hyperpolarized [1-(13)C]pyruvate through lactate dehydrogenase (LDH) activity. In GS-2 glioblastoma cells, PI3K inhibition by LY294002 or everolimus caused hyperpolarized lactate to drop to 42 +/- 12% and to 76 +/- 5%, respectively. In MDA-MB-231 breast cancer cells, hyperpolarized lactate dropped to 71 +/- 15% after treatment with LY294002. These reductions were correlated with reductions in LDH activity to 48 +/- 4%, 63 +/- 4%, and 69 +/- 12%, respectively, and were associated with a drop in levels of LDHA mRNA and LDHA and hypoxia-inducible factor-1alpha proteins. Supporting these findings, tumor growth inhibition achieved by everolimus in murine GS-2 xenografts was associated with a drop in the hyperpolarized lactate-to-pyruvate ratio detected by in vivo MRS imaging, whereas an increase in this ratio occurred with tumor growth in control animals. Taken together, our findings illustrate the application of hyperpolarized (13)C MRS of pyruvate to monitor alterations in LDHA activity and expression caused by PI3K pathway inhibition, showing the potential of this method for noninvasive imaging of drug target modulation.

Figure 1

Effect of LY294002 on PI3K signaling in GS-2 cells. A, Western blots showing depletion of phospho-4E-BP1 levels in GS-2 following a 48-hr exposure to 50 μM LY294002. Total 4E-BP1 is shown as a loading control. B, WST-1 cell proliferation assay showing a reduction in the proliferation rate over 48 hrs.

Figure 2

Conversion of hyperpolarized [1-¹³C]-pyruvate to [1-¹³C]-lactate in a GS-2 perfused cell experiment. A, Representative ¹³C spectrum 45 sec after the addition of hyperpolarized [1-¹³C]-pyruvate (final concentration of 1 mM). Resonances for pyruvate, pyruvate hydrate and lactate are indicated. B, Peak areas of hyperpolarized pyruvate (note that the pyruvate peak areas were divided by 4) and lactate during acquisition of 1 mM hyperpolarized pyruvate injection.

Figure 3

Effect of PI3K inhibition by LY294002 on hyperpolarized lactate formation in GS-2 perfused cells. A, Evolution of [1-¹³C]-lactate peak areas after addition of 1 mM hyperpolarized [1-¹³C]-pyruvate to control or LY294002-treated perfused GS-2 cells, showing reduction in hyperpolarized lactate formation with treatment. Continuous lines represent the fits to Bloch equations. B, Reproducible reductions in maximum hyperpolarized lactate (Lac_max) levels in response to treatment over several concentrations of hyperpolarized pyruvate, indicating that the use of Lac_max to probe the effect of PI3K inhibition is reproducible independent of pyruvate concentration. C, Plot of k_Pyr versus Lac_max, indicating a correlation between the two methods of hyperpolarized ¹³C data analysis. Line, best linear fit (R²=0.93).

Figure 4

Effect of PI3K inhibition on endogenous metabolites detected by ³¹P MRS. ³¹P MR spectrum obtained from perfused GS-2 cells. Metabolites detectable in spectrum: PE, phosphoethanolamine; PC, phosphocholine; P_{i ex}, extracellular inorganic phosphate; P_{i in}, intracellular inorganic phosphate; GPE, glycerophosphoethanolamine; GPC, glycerophosphocholine; PCr, phosphocreatine; α-NTP, β-NTP and γ-NTP, nucleotide triphosphates; UDPS, UDP-sugars. Inset, expansion of the phosphomonoester and phosphodiester region (0 – 5 ppm) from control and LY294002-treated cells, indicating a drop in PC and PE in LY294002-treated cells.

Figure 5

Effect of PI3K inhibition by LY294002 on LDH activity, LDH levels and HIF-1αlevels in GS-2 and MDA-MB-231 cells. A, V_max of LDH activity in cell lysates, showing a decrease in the activity with PI3K inhibition. B, Western blot analysis revealing decreases in levels of LDHA and HIF-1αafter treatment. GAPDH is shown as a loading control. C, RT-PCR analysis showing a decrease in LDHA gene expression following PI3K inhibition.

Figure 6

Effect of everolimus treatment on GS-2 tumor xenografts. A, coronal image (7 mm from the surface coil) overlaid with tumor voxels (top) illustrating the origin of 2D-MRSI spectra (middle) and overlay of relative lactate-to-pyruvate ratio maps (bottom) prior to (left) and following 2 days of treatment (right). B, Tumor volume in control (continuous lines) and treated (dotted lines) tumors. C, Lactate-to-pyruvate ratio in control (continuous lines) and treated (dotted lines) tumors.