The phosphoinositide 3-kinase inhibitor PI-103 downregulates choline kinase alpha leading to phosphocholine and total choline decrease detected by magnetic resonance spectroscopy

Abstract

The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.

Figure 1

Metabolic changes caused by PI-103 in human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cell lines. A, representative in vitro ³¹P-MRS and expansion of the choline-containing metabolites region of ¹H-MR spectra of PC3 aqueous cell extracts following treatment with PI-103 (5× GI₅₀, 500 nmol/L) compared with vehicle-treated (DMSO) control; Gluc, glucose. B, representative in vitro ³¹P-MRS and expansion of the choline-containing metabolites region of ¹H-MR spectra of HCT116 aqueous cell extracts following treatment with PI-103 (5× GI₅₀, 5 μmol/L) compared with vehicle-treated (DMSO) control. C, comparison of cellular effects and ³¹P-MRS–detected metabolic changes caused by PI-103 (5× GI₅₀) in PC3 prostate cancer and HCT116 colon cancer cells at 24 h. Results are expressed as % treated versus control (% T/C); columns, mean of at least three separate experiments; bars, SD. Statistically significantly different from the control; *, P ≤ 0.05; **, P < 0.01; †, P ≤ 0.005; and ‡, P < 0.0005; two-tailed unpaired t test was used for all comparisons.

Figure 2

Molecular effects of inhibition with PI-103 on the PI3K signaling pathway in PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cell lines. A, representative Western blots showing decreases in molecular biomarkers in the PI3K signaling pathway in PC3 prostate cancer cells following treatment with PI-103 at 1×, 2.5×, and 5× GI₅₀, 24 h posttreatment. B, representative Western blots showing decreases in molecular biomarkers in the PI3K signaling pathway in PC3 prostate cancer cells at selected time points posttreatment with PI-103 (5× GI₅₀). C, representative Western blots showing inhibition of molecular biomarkers in the PI3K signaling pathway and changes in protein expression levels of enzymes involved in lipid metabolism in HCT116 colon cancer cells following treatment with PI-103 (5× GI₅₀).

Figure 3

Effects of inhibition with PI-103 on cell cycle distribution in PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cell lines. A, representative flow cytometry analysis histograms showing the cell cycle distribution of PC3 cells with vehicle treatment (DMSO, control, solid) or following treatment with PI-103 at 1×, 2.5×, and 5× GI₅₀, 24 h posttreatment (empty). B, representative flow cytometry analysis histograms showing the cell cycle distribution of PC3 cells with vehicle treatment (DMSO, control, solid) or following treatment with PI-103 (5× GI₅₀, empty) at selected time points posttreatment. C, representative flow cytometry analysis histograms showing the cell cycle distribution of HCT116 cells with vehicle treatment (DMSO, control, black) or following treatment with PI-103 (grey).

Figure 4

Investigation of mechanisms underlying MRS-detected changes in PC levels following treatment of PTEN null human prostate cancer cells PC3 with PI-103. A, comparison of cellular effects and ¹H-MRS–detected metabolic changes caused by PI-103 and docetaxel (DTX) in PC3 cells at 5× GI₅₀, 24 h. B, representative Western blots showing changes in protein expression levels of FAS, pACL (Ser454), and ChoK_α at selected time points posttreatment with PI-103 (5× GI₅₀). C, changes in PC (³¹P-MRS) normalized to cell volume and ChoK_α protein expression levels (densitometry) in PC3 cells over the time course of treatment with PI-103 (5× GI₅₀). Results are expressed as % T/C; points, mean of at least three separate experiments; bars, SD. Statistically significantly different from the control; *, P ≤ 0.05; **, P < 0.01; †, P ≤ 0.005; ‡, P < 0.0005; two-tailed unpaired t test was used for all comparisons.