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Logo of neuroncolAboutAuthor GuidelinesEditorial BoardNeuro-Oncology
 
Neuro Oncol. Mar 2012; 14(3): 315–325.
Published online Dec 12, 2011. doi:  10.1093/neuonc/nor209
PMCID: PMC3280799
Reduced phosphocholine and hyperpolarized lactate provide magnetic resonance biomarkers of PI3K/Akt/mTOR inhibition in glioblastoma
Humsa S. Venkatesh, Myriam M. Chaumeil, Christopher S. Ward, Daphne A. Haas-Kogan, C. David James, and Sabrina M. Ronen
Department of Radiology and Biomedical Imaging (H.S.V., M.M.C., C.S.W., S.M.R.) and Department of Neurological Surgery, Brain Tumor Research Center, University of California San Francisco, California (D.A.H.-K., C.D.J.)
Corresponding Author: Sabrina M. Ronen, PhD, 1700 4th St., Box 2532, Byers Hall 3rd Floor, Suite, University of California, San Francisco, San Francisco, CA 94158 (sabrina.ronen/at/ucsf.edu).
Received August 23, 2011; Accepted October 28, 2011.
The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is activated in more than88% of glioblastomas (GBM). New drugs targeting this pathway are currently in clinical trials. However, noninvasive assessment of treatment response remains challenging. By using magnetic resonance spectroscopy (MRS), PI3K/Akt/mTOR pathway inhibition was monitored in 3 GBM cell lines (GS-2, GBM8, and GBM6; each with a distinct pathway activating mutation) through the measurement of 2 mechanistically linked MR biomarkers: phosphocholine (PC) and hyperpolarized lactate.31P MRS studies showed that treatment with the PI3K inhibitor LY294002 induced significant decreases in PC to 34 %± 9% of control in GS-2 cells, 48% ± 5% in GBM8, and 45% ± 4% in GBM6. The mTOR inhibitor everolimus also induced a significant decrease in PC to 62% ± 14%, 57% ± 1%, and 58% ± 1% in GS-2, GBM8, and GBM6 cells, respectively. Using hyperpolarized 13C MRS, we demonstrated that hyperpolarized lactate levels were significantly decreased following PI3K/Akt/mTOR pathway inhibition in all 3 cell lines to 51% ± 10%, 62% ± 3%, and 58% ± 2% of control with LY294002 and 72% ± 3%, 61% ± 2%, and 66% ± 3% of control with everolimus in GS-2, GBM8, and GBM6 cells, respectively. These effects were mediated by decreases in the activity and expression of choline kinase α and lactate dehydrogenase, which respectively control PC and lactate production downstream of HIF-1. Treatment with the DNA damaging agent temozolomide did not have an effect on either biomarker in any cell line. This study highlights the potential of PC and hyperpolarized lactate as noninvasive MR biomarkers of response to targeted inhibitors in GBM.
Keywords: glioblastoma (GBM), hyperpolarized lactate, magnetic resonance spectroscopy (MRS), phosphocholine (PC), PI3K/Akt/mTOR pathway
The overall incidence of primary brain and central nervous system tumors is ~7 per 100,000 persons per year in the United States.1 Glioblastoma (GBM) is the most common (36% of total) and malignant type of primary brain tumor in young adults. Standard clinical care for patients with GBM currently consists of surgical resection followed by treatment with radiation and chemotherapy.2 Nonetheless, survival rates are only ~15 months,3 and alternative therapies are critically needed to improve patient survival. As the specific molecular events associated with the disease are identified and characterized, researchers have begun to focus on the potential of molecularly targeted treatments that specifically inhibit oncogenic events.
The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is an important therapeutic target in cancer treatment. This signaling pathway is vital for regulation of several cell processes, including cell growth, proliferation, and survival.4 In the case of GBM, genome-wide profiling studies have shown that 88% of cases harbor at least 1 mutation leading to constitutive activation of the PI3K/Akt/mTOR pathway, thus highlighting the potential for inhibiting this pathway as an effective treatment for these tumors.5 Several clinical trials assessing the therapeutic efficacy of inhibitors of the PI3K/Akt/mTOR pathway, either alone or in combination with traditional therapies, are in fact under way in GBM (http://clinicaltrials.gov/). However, the effect of this inhibition is not necessarily easy to detect using anatomical imaging techniques, because response can be associated with tumor stasis or growth inhibition rather than shrinkage.6,7 To improve patient treatment and outcomes, it is therefore important to identify and validate alternative approaches that can be used to inform on drug delivery and efficacy.
The recent surge in research on cancer metabolism is elucidating the link between deregulated signaling pathways and altered cellular metabolism. Accordingly, changes in cell metabolism can serve as biomarkers of the therapeutic efficacy of drugs that target signaling. Magnetic resonance spectroscopy (MRS) provides a powerful tool to probe cellular metabolism. Combining 31P, 1H, and 13C MRS, it is possible to noninvasively study endogenous metabolite levels and specific metabolic fluxes within cells and tissues.813
Using 1H MRS in an orthotopic GBM model, we have previously shown that the inhibition of PI3K using PX-866 results in a decrease in total choline-containing metabolite levels (tCho), composed of choline (Cho), phosphocholine (PC), and glycerophosphocholine (GPC).14 Consistent with this finding, 31P MRS has been used for detecting decreased PC levels in various cancer types following treatment with LY294002.15,16 More recently, we have monitored the conversion of pyruvate to lactate using hyperpolarized 13C MRS, a novel method that provides enhancement in signal-to-noise ratio (SNR) of over 10 000-fold when compared with traditional 13C MRS.17 We found that hyperpolarized lactate produced from exogenous hyperpolarized pyruvate decreases following inhibition of the PI3K/Akt/mTOR pathway and, thus, can also be used as an indicator of drug target modulation.1820
Signaling via the PI3K/Akt/mTOR pathway controls the expression of several transcription factors, including the hypoxia inducible factor 1α (HIF-1α). HIF-1 regulates the expression of multiple enzymes.21,22 Most notably, it controls the expression of choline kinase alpha (ChoKα),23 an isoform of ChoK that is the enzyme that catalyzes the synthesis of PC from Cho, and the expression of lactate dehydrogenase-A (LDHA), the dominant isoform of LDH that catalyzes the conversion of pyruvate into lactate.24,25 We therefore hypothesized that inhibition of the PI3K/Akt/mTOR pathway would lead to a decrease in the expression of both ChoKα and LDHA, resulting in a decrease in the levels of both PC and hyperpolarized lactate.
To test this hypothesis, we used MRS to study the effect of PI3K and mTOR inhibition in 3 GBM cell lines, each with a different activating mutation in the PI3K/Akt/mTOR pathway. Our investigation confirmed that signal inhibition results in decreased expression and activity of ChoK and LDH and highlights the value of PC and hyperpolarized lactate as downstream metabolic biomarkers of response to PI3K/Akt/mTOR inhibitors in GBM, independent of the genetic basis for pathway activation.
Cell Culture
GS-2, GBM8, and GBM6 cells26,27 were supplied by the University of California, San Francisco, Brain Tumor Research Center Preclinical Therapeutics Core. Fingerprints have been established for the cell lines and used to confirm their identities.27,28
Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin, 1 mM sodium pyruvate, and 28 mM glucose. Custom-made DMEM with 0.22 g/L inorganic phosphate (Pi; UCSF Cell Culture Facility) was used in MRS studies.
For PI3K inhibition, cells were incubated with 50 µM LY294002 (LC Laboratories). For mTOR inhibition, cells were treated with 100 nM everolimus (Molcan Corporation). To investigate the effect of a DNA damaging agent, cells were treated with 100 μM temozolomide (Tecoland Corp). The final concentration of DMSO used to dissolve all inhibitors was 1:1000 in culture medium. For all studies the effect of treatment was monitored at 48 h.
Western Blot Analysis
Phospho-4E-BP1 (P-4E-BP1) was used as a downstream reporter of PI3K and mTOR inhibition, carbonic anhydrase IX (CA-IX) as a downstream reporter of HIF-1α inhibition, and β-actin as a loading control. Control and treated cells were lysed using Cell Lysis Buffer (Cell Signaling). Lysates normalized to cell number were then run on 4% to 20% gels (Bio-Rad) by the SDS-PAGE method and electrotransferred onto nitrocellulose membranes. Blots were blocked and incubated with the primary antibodies anti-4E-BP1 (Cell Signaling), anti-P-4E-BP1 (Ser 65, Cell Signaling), anti-β-actin (Cell Signaling), anti-LDHA (Epitomics), or anti-CA-IX (Abcam), then incubated with secondary antibody anti-IgG HRP-linked antibody (Cell Signaling). Immunocomplexes were visualized using ECL Western Blotting Substrate (Pierce).
Cell Proliferation Assay
Cell proliferation in response to drug treatment was assessed by manual cell counts 48 h after treatment and was further confirmed by using the WST-1 cell proliferation assay (Roche). For the WST-1 assay, cells were seeded in 96-well plates (1 × 104 cells per well in 200 μL of culture medium) and treated with either LY294002, everolimus, or temozolomide for 48 h. Cells were then incubated for 1 h with WST-1 reagent and viability quantified based on the absorbance at 440 nm (Infinite M200; Tecan).
ChoK Activity Assay
ChoK activity assays were performed as previously described.29,30 In brief, cell lysates were prepared in 550 μL of cell lysis buffer and combined with reactants in a 5-mm NMR tube to final concentrations of 5 mM Cho, 10 mM ATP, and 10 mM MgCl2 in Tris-HCl buffer. An array of 1H NMR spectra was acquired on a 600 MHz NMR spectrometer (Varian) with a 90° pulse, a 3-s relaxation delay for a total acquisition time of 1 h. Evolution of Cho and PC was monitored at 25°C. Spectra were integrated using ACD/Spec Manager and normalized to external TMS reference (5 mM). The linear regression of the buildup of PC was used to calculate ChoK activity in mmol/min.
LDH Activity Assay
The enzymatic activity of LDH was measured in fresh cell lysates by monitoring NADH consumption after addition of varying concentrations of pyruvate, as previously described.20,31 Using an Infinite M200 spectrophotometer (Tecan), NADH levels were monitored based on absorbance values at 340 nm over a span of 10 min. Lineweaver-Burke plot analysis was used to determine KM and Vmax values.
Gene Expression Analysis
Total cellular RNA was extracted using an RNeasy Mini kit (Qiagen). Total RNA was quantified using a Nano-Drop ND1000 Fluorospectrometer (NanoDrop Technologies). The QuantiTect Reverse Transcription kit (Qiagen) was used for reverse transcription. Real-time polymerase chain reaction (RT-PCR) of resulting cDNA was performed on a Taqman 7900 (Applied Biosystems). Expression levels were quantified with gene-specific primer/probe sets (Applied Biosystems) specific for human LDHA (Hs00855332_g1), CAIX (Hs00154208_m1), CHKA (Hs03682798_m1), and CHKB (Hs00193219_m1). Data were normalized to the housekeeping gene ACTB (Integrated DNA Technologies).
Perfused Cell System Setup
For MRS studies of live cells, 1.5–2 × 108 cells were encapsulated in agarose beads, as previously described.20,32 After overnight incubation, beads were loaded into a 10-mm NMR tube connected to a perfusion system modified from that previously described.20,32 In brief, the perfusion system circulated medium throughout the tube at a constant flow of 1.5 mL/min, a separate tube being used to deliver 5% CO2/95% air. A port on the inflow line allowed for injection of hyperpolarized material, during which perfusion was briefly stopped. The NMR tube was maintained at 35°C throughout all MRS studies.
31P MRS Acquisition and Analysis
31P MRS spectra were acquired on a 500-MHz INOVA spectrometer (Varian) with a 30° pulse, 3-second repetition time, and composite pulse proton decoupling during acquisition. The resulting spectra were analyzed using ACD/Spec Manager, version 9.15 (Advanced Chemistry Development). After deconvolution, metabolite concentrations were calculated from peak areas and normalized to both cell number and internal reference (medium Pi, 1.87 µM).
Hyperpolarization
For hyperpolarization studies, ~6 μL [1-13C]-pyruvic acid (Isotec) containing 15 mM of the trityl radical OX063 (Oxford Instruments) was hyperpolarized using a Hypersense DNP (Oxford Instruments) polarizer as described elsewhere.33,34 After an hour, hyperpolarized pyruvate was dissolved in 6.0 mL of isotonic 40 mM Tris-based buffer containing 3.0 µM EDTA (pH 7.8) and injected into the perfusion system. The final concentration of hyperpolarized material inside the sample was 5 mM.
13C MRS Acquisition and Analysis
Dynamic sets of HP 13C spectra were acquired with 13° excitation pulses and a 3-s repetition time for a total of 300 s. The resulting spectra were quantified by peak integration using ACD/Spec Manager. To correct for potential variations in the degree of polarization, peak areas of hyperpolarized species were normalized to the total hyperpolarized signal at maximum pyruvate value. All signals were also normalized to cell number. Maximum hyperpolarized lactate levels per cell were determined as an indicator of the extent of hyperpolarized lactate production from hyperpolarized pyruvate.20
Statistical Analysis
All results, expressed as mean ± standard deviation, represent a mean of at least 3 repeats, unless otherwise specified. Two-tailed unpaired Student's t test was used to establish the statistical significance of differences, with P ≤ .05 considered to be statistically significant.
In this investigation, we looked at the effects of PI3K/Akt/mTOR pathway inhibition using 3 GBM cell lines. We investigated GS-2 cells, in which the pathway is activated through loss of PTEN, GBM8, in which EGFR is amplified (PTEN is wild-type) and GBM6, in which the pathway is activated through EGFR mutation and amplification (PTEN is wild-type).26,27 Combined, the 3 cell lines provide representation of gene alterations found in the majority of GBM tumors. The effect of the prototype PI3K inhibitor LY294002 and the clinically relevant mTOR inhibitor everolimus were investigated. As a control, we also monitored the effect of the clinically relevant DNA damaging agent temozolomide, which is not expected to affect PI3K/Akt/mTOR signaling.
PI3K/Akt/mTOR Inhibition Leads to Reduced P-4E-BP1 Expression, Reduced HIF-1 Transcriptional Activity, and Inhibition in Cell Proliferation
Western blot analysis for P-4E-BP1 confirmed that treatment with LY294002 or everolimus resulted in inhibition of signaling via the PI3K/Akt/mTOR pathway. In all 3 cell lines, P-4E-BP1 levels decreased, whereas total 4E-BP1 levels remained unchanged. In contrast, temozolomide treatment did not affect P-4E-BP1 levels, confirming the fact that this drug does not affect PI3K/Akt/mTOR signaling (Fig. 1).
Fig. 1.
Fig. 1.
Western blot analysis of P-4EBP1, total-4EBP1, and CA-IX in GS-2, GBM6, and GBM8 cell lines treated with temozolomide, LY294002, and everolimus. In all 3 cell lines, P-4EBP1 and CA-IX protein levels were lower following treatment with everolimus and LY294002, (more ...)
To evaluate the downstream consequences of PI3K/Akt/mTOR inhibition and, in particular, the effect on HIF-1 transcriptional activity, we probed for CA-IX, a sensitive downstream reporter of HIF-1. Western blotting showed substantial decreases in protein levels in all 3 cell lines following both LY294002 and everolimus treatment (Fig. 1). In addition, RT-PCR studies were performed to address the effect of treatments on mRNA levels of CA-IX. As shown in Fig. 2, both LY294002 and everolimus treatments caused a significant decrease in CA-IX mRNA in each GBM cell line. On average, LY294002 and everolimus treatments induced a significant decrease to 35% ± 25% (P = 5 × 10−5) and 58% ± 14% (P = 2 × 10−5) of control, respectively. As expected, temozolomide had no effect on CA-IX levels or expression (P = .8).
Fig. 2.
Fig. 2.
RT-PCR analysis of GS-2, GBM8, and GBM6 cell lines treated with temozolomide, LY294002, and everolimus. CA-IX mRNA expression was significantly decreased following LY294002 treatment in each cell line: 10% ± 2% of control in GS-2 (P = 2 × (more ...)
The effect of each treatment on cell proliferation was then assessed by manual cell count at 48 h. All 3 treatments induced a significant inhibition of cell proliferation in each cell line (P < .05 for all 3 cell lines compared with control). LY294002 inhibited cell growth to 53% ± 5%, 64% ± 5%, and 63% ± 7% of control in GS-2, GBM8, and GBM6 cells, respectively. Similarly, everolimus led to inhibition of growth to 53% ± 6%, 64% ± 5%, and 63% ± 3%. Temozolomide caused decreases to 59% ± 6%, 65% ± 4%, and 60% ± 4%, respectively (data not shown). Results from the WST-1 assay at 48 h were comparable within experimental error.
PI3K/Akt/mTOR Inhibition Leads to a Decrease in ChoKα Expression, ChoK Activity, and PC Levels
Table 1 summarizes the effect of treatment on ChoKα expression, as determined by RT-PCR. In all 3 cell lines, a significant decrease in ChoKα mRNA levels was observed following treatment with either the PI3K inhibitor or the mTOR inhibitor. On average, ChoKα mRNA levels decreased to 58% ± 11% of control in LY294002-treated cells (P = .02) and to 47% ± 3% in everolimus-treated cells (P = .001). In contrast, temozolomide did not induce any significant change in the expression of ChoKα (P = .5). Levels of ChoKβ (the second isoform of ChoK) remained unchanged in every case (data not shown).
Table 1.
Table 1.
ChoKα mRNA, ChoK activity, and PC levels expressed as percentage control for GS-2, GBM8, and GBM6 cell lines treated with LY294002, everolimus, and temozolomide
Next, to confirm the mRNA findings, the cellular activity of ChoK was measured using 1H MRS by monitoring the real-time kinetics of PC production in cell lysates.29,35 Consistent with the decrease in ChoKα mRNA levels, a significant decrease in the rate of PC production was observed following treatment with LY294002 and everolimus (Table 1). As illustrated in Fig. 3A, control GS-2 cells produced PC at a rate of 8.4 ± 0.2 fmol/cell/h, whereas LY294002 and everolimus decreased production to 4.7 ± 0.4 fmol/cell/h and 5.0 ± 1.0 fmol/cell/h, respectively. Similarly, in GBM8 cells, PC production rates decreased from 14.6 ± 0.3 fmol/cell/h in control cells to 9.7 ± 1.0 fmol/cell/h in LY294002-treated cells and to 8.5 ± 1.3 fmol/cell/h in everolimus-treated cells. Finally, in GBM6, PC production rates were 3.8 ± 0.3, 1.8 ± 0.5, and 2.5 ± 0.1 fmol/cell/h in control, LY294002-treated, and everolimus-treated cells, respectively. Temozolomide treatment did not induce any significant change in ChoK activity in any of the 3 cell lines.
Fig. 3.
Fig. 3.
(A) PC level as measured by 1H MRS in cell lysates as a function of time for control GS-2 cells and GS-2 cells treated with LY294002, everolimus, and temozolomide ( 3 repeats per treatment). The rate of PC production was 8.4 ± 0.2 fmol/cell/h (more ...)
Finally, 31P MRS of live perfused GBM cells was performed to assess the effect of PI3K/Akt/mTOR inhibition on PC levels. Figure 3B presents data obtained from the GS-2 cell line and illustrates the decrease in PC levels with LY294002 treatment, compared with control. As summarized in Table 1, treatment with LY294002 induced a significant decrease in PC levels in all 3 cell lines: from 1.33 ± 0.32 fmol/cell to 0.45 ± 0.33 fmol/cell in GS-2 cells, from 1.31 ± 0.15 to 0.63 ± 0.1 fmol/cell in GBM8, and from 1.57 ± 0.12 to 0.73 ± 0.07 fmol/cell in GBM6 cells. Everolimus caused a significant decrease in PC levels to 0.82 ± 0.32, 0.74 ± 0.02, and 0.91 ± 0.16 fmol/cell in GS-2, GBM8, and GBM6 cells, respectively. In contrast, all 3 cell lines treated with temozolomide showed no change in PC levels (Table 1).
PI3K/Akt/mTOR Inhibition Leads to a Decrease in LDH Expression and Activity and Conversion of Hyperpolarized Pyruvate to Lactate
Western blot analysis indicated that LDHA protein levels dramatically decreased after inhibition of the PI3K/Akt/mTOR pathway with LY294002 or everolimus in GS-2, GBM8, and GBM6 cells (Fig. 4). RT-PCR was performed to quantitatively address the effect of treatment on LDHA mRNA expression. Consistent with the decrease in protein level, LDHA expression decreased on average for all cell lines to 59% ± 6% following treatment with LY294002 (P = .007), and everolimus caused a decrease to 68% ± 6% of control cells (P = .01) (Table 2). Temozolomide had no effect on either LDHA protein levels or mRNA expression (P = .9).
Fig. 4.
Fig. 4.
Western blot analysis of LDHA in GS-2, GBM8, and GBM6 cell lines treated with temozolomide, LY294002, and everolimus. In all 3 cell lines, LDHA protein levels were lower following treatment with everolimus and LY294002, in line with the reduced transcriptional (more ...)
Table 2.
Table 2.
LDHA mRNA, LDH activity, and maximum hyperpolarized lactate level (HP lactate) expressed as percentage control for GS-2, GBM8, and GBM6 cell lines treated with LY294002, everolimus, and temozolomide
The activity of LDH was then examined using a spectrophotometric method. Consistent with the decrease in protein levels, cellular Vmax levels decreased in all 3 cell lines with inhibition of the PI3K/Akt/mTOR pathway, as presented in Table 2. In control cells, Vmax values were 21.4 ± 0.3, 13.5 ± 0.7, and 14.2 ± 0.8 μmol NADH/min per 107 cells for GS-2, GBM8, and GBM6 cells, respectively. When treated with LY294002, Vmax decreased to 10.4 ± 0.2, 8.5 ± 0.4, and 6.9 ± 0.1 μmol NADH/min per 107 cells, and everolimus caused decreases to 13.6 ± 0.5, 8.2 ± 0.6, and 8.4 ± 0.9 μmol NADH/min per 107 cells in GS-2, GBM8, and GBM6 cells, respectively. Temozolomide had no effect on LDH activity.
To study the metabolic consequences of LDH inhibition and to confirm that MRS can be used as a method to monitor this inhibition, we investigated the conversion of hyperpolarized pyruvate into lactate using dynamic hyperpolarized 13C MR spectroscopy. After exposing live cells to hyperpolarized labeled [1-13C] pyruvate, the build-up of labeled lactate was monitored over the span of 5 min. Figure 5 shows 2 hyperpolarized 13C MR data sets obtained from perfused GBM8 cells and the decrease in hyperpolarized lactate production following everolimus treatment. Figure 6 presents the evolution of hyperpolarized lactate levels versus time for the GS-2 cell line, with results confirming the efficient conversion of hyperpolarized pyruvate into lactate in every case and further demonstrating the decrease in hyperpolarized lactate levels following PI3K/Akt/mTOR signal inhibition, but not following temozolomide treatment. Table 2 summarizes the results obtained for all 3 cell lines and all 3 treatments. Maximum hyperpolarized lactate levels were significantly decreased following PI3K/Akt/mTOR inhibition by LY294002 or everolimus in GS-2, GBM8, and GBM6 cells, whereas temozolomide did not affect lactate production.
Fig. 5.
Fig. 5.
Stack plots of hyperpolarized 13C MR spectra acquired after injection of exogenous hyperpolarized pyruvate in perfused GBM8 control cells (A) and cells treated for 48 h with everolimus (B). The resonances of pyruvate (173 ppm), pyruvate hydrate (181 ppm), (more ...)
Fig. 6.
Fig. 6.
Hyperpolarized lactate intensity as a function of time as assessed from HP 13C MR spectra of control, temozolomide-treated, everolimus-treated, and LY294002-treated perfused GS-2 cells. Both LY294002 and everolimus induced a significant decrease in the (more ...)
Because of the high prevalence of PI3K/Akt/mTOR pathway activation in GBM tumors,5 targeted therapies that specifically inhibit this signaling pathway are of high interest for incorporating into the treatment of patients with GBM. However, evaluation of response to such molecularly targeted treatments remains a challenge, because conventional imaging methods are often limited to the detection of anatomical changes. As a consequence, new metabolic biomarkers that inform on drug delivery and molecular target inhibition are critically needed. In this study, we evaluated the potential of 2 mechanistically linked biomarkers: PC as assessed by 31P MRS and hyperpolarized lactate as assessed by hyperpolarized 13C MRS.
To mimic the type of treatments currently in clinical trials and to address the generality of our observations, we monitored the effect of 2 inhibitors of the PI3K/Akt/mTOR pathway: the clinically relevant mTOR inhibitor everolimus and the PI3K inhibitor LY294002. Studies were performed in 3 GBM cells lines with distinct signature gene alterations, each of which leads to activation of the PI3K/Akt/mTOR pathway. Accordingly, on the basis of the recently published study from The Cancer Genome Atlas Research Network,5 the 3 cell lines studied collectively recapitulate the genetic make-up that would be expected in the majority of patients with GBM with regard to the PI3K/Akt/mTOR pathway.
Following signal inhibition, we observed a decrease in signaling downstream of mTOR and a decrease in HIF-1 transcriptional activity in all cell lines and with either treatment. These results are consistent with previous studies demonstrating a decrease in the translation of HIF-1α following inhibition of the PI3K signaling cascade.36,37 Our results are also consistent with previous reports of decreased HIF-1α levels in several cancer models following treatment with everolimus38 or LY294002.39
HIF-1 is known to regulate the expression of ChoKα23 and several glycolytic enzymes, including LDHA.24,25 We confirmed that the decrease in HIF-1 transcriptional activity following inhibition of the PI3K/Akt/mTOR pathway in our cell models was associated with a decrease in expression and activity of ChoK and LDH. Furthermore, the decrease in enzyme activities explains the reduction in PC and in hyperpolarized lactate that was observed in our cell lines following treatment with the PI3K or mTOR inhibitors. These observations are in line with a previous study demonstrating a decrease in PC following inhibition of HIF-1α in a colorectal cancer model.40 These findings also indicate that the 2 metabolic biomarkers, PC and hyperpolarized lactate, are mechanistically linked and can serve as complementary readouts of PI3K/Akt/mTOR signal inhibition upstream of HIF-1α.
Whereas HIF-1α expression is dependent on PI3K/Akt/mTOR signaling, the stability of HIF-1α depends on oxygen levels, and HIF-1α levels are typically higher under hypoxic conditions. Because our cell studies were performed under normoxic conditions (95% air/5% CO2), it could be argued that our observations in cells will be obfuscated in the in vivo setting, wherein tumor cells are frequently hypoxic. However, in a parallel study, we recently showed that the observations described here are also applicable to the in vivo setting, at least for one of the cell lines investigated in this study.18 Specifically, we studied the effect of everolimus on GS-2 orthotopic GBM tumors in rats and demonstrated a significant decrease in the lactate-to-pyruvate ratio in treated animals when compared with controls. Furthermore, we demonstrated that this metabolic effect was mediated in vivo by a decrease in the transcriptional activity of HIF-1 and a decrease in LDHA expression, similar to findings in the current cell study. Of significance, the decrease in hyperpolarized lactate occurred in vivo over a week earlier than the effect on tumor size detected by standard MR anatomical imaging. Our finding that PC levels decrease following PI3K/Akt/mTOR signal inhibition is also consistent with our previous work in GBM tumors in which we observed a decrease in tCho using 1H MRS in orthotopic U87 tumors.14 The drop in tCho is likely to have been a result of a decrease in PC, which is typically the main component of tCho in tumors.
As a control, we monitored the effect of temozolomide, a DNA-damaging agent commonly used to treat GBM2 but not expected to affect the PI3K/Akt/mTOR signaling pathway. Our results confirm that, in the cell lines examined, temozolomide treatment had no effects on P-4EBP1, CA-IX, and LDHA expression. Consistent with this, no differences in the levels of PC and hyperpolarized lactate were observed between control cells and temozolomide-treated cells for all 3 cell lines. Nonetheless, it should be noted that we have recently performed an in vivo study aimed at monitoring the effect of temozolomide in U87 orthotopic brain tumors in rats. In that study, a decrease in the pyruvate-to-lactate conversion following treatment was observed,41 and we have subsequently shown that this effect is specific to tumors that respond to treatment, while not being observed in tumors that express active O-6-methylguanine-DNA methyltransferase (MGMT), and therefore, do no respond to temozolomide.42 The underlying mechanism for this effect remains to be elucidated, but we have shown that it is not associated with a decrease in LDHA expression. A decrease in hyperpolarized lactate has also been observed in C6 gliomas following radiotherapy.43 Thus, whereas the drop in hyperpolarized lactate following signaling inhibition is not a specific metabolic effect associated with PI3K/Akt/mTOR inhibition, it nonetheless provides an informative biomarker of drug delivery and pathway inhibition by such targeted therapeutics.
From a clinical perspective, 1H MRS is the spectroscopic method most widely implemented on clinical systems. It can be used to detect both tCho and lactate levels. As mentioned above, because tCho is comprised of Cho, PC, and GPC and because PC is typically the largest component of the tCho peak in cancer cells, 1H MRS is likely to be as useful as 31P MRS for detecting a decrease in PC and, thus, informing on the effect of treatment. However, although intrinsically less sensitive, 31P MRS remains the method of choice for resolving phosphorylated compounds, such as PC, and approaches such as 1H to 31P polarization transfer have recently been developed to increase detection sensitivity at clinical field strength for potential use in clinical examinations.44
With regard to detection of lactate by 1H MRS, this biomarker has been used in vivo for the diagnosis and evaluation of treatment response in preclinical and clinical settings, including with GBM.45,46 However, lactate detection by 1H MRS presents several limitations. The lactate resonance overlaps with resonances of cerebral lipids, and the implementation of special editing methods is necessary to accurately measure lactate levels in vivo.47 Furthermore, the lactate level measured by 1H MRS represents the total lactate pool, which is composed of both metabolically active and inactive lactate. Metabolically active lactate, which is lactate produced from pyruvate by live cells, can inform on the effects of treatment. The inactive lactate represents the pool of extracellular lactate, which is known to frequently accumulate in poorly vascularized necrotic regions and, thus, cannot reliably inform on response to treatment.48 In contrast to 1H MRS, hyperpolarized 13C MRS measures hyperpolarized lactate formed from exogenous hyperpolarized pyruvate. It therefore informs on the metabolism of live cells and can serve as a metabolic biomarker of treatment effects. Over the past few years, hyperpolarized 13C MRS has shown tremendous potential for the diagnosis and follow-up of several types of cancer in vivo in preclinical models.13,1820,33,49 This method is now in clinical trial in patients with prostate cancer at University of California, San Francisco (http://clinicaltrial.gov) and is generating very promising results. Moving forward, this new imaging approach is therefore likely to be applied clinically to other cancer types and could serve as a noninvasive approach for monitoring response to novel PI3K/Akt/mTOR inhibitors.
In summary, we reported here that inhibition of the PI3K/Akt/mTOR signaling pathway can be monitored in GBM cells through the assessment of 2 noninvasive mechanistically linked metabolic MR biomarkers: PC and hyperpolarized lactate. Considering that the PI3K/Akt/mTOR pathway is activated in more than88% of GBM, such findings highlight the potential of MRS as a noninvasive imaging approach for informing on drug delivery and drug target modulation in patients with GBM who are treated with drugs that inhibit this pathway.
Funding
This work was supported by National Institutes of Health (NIH) UCSF Brain Tumor SPORE (P50 CA097257); UC Discovery grants in conjunction with GE Healthcare; NIH (RO1 CA130819); and NIH (RO1 CA154915).
Acknowledgments
We thank Alessia Lodi for help with gene expression analysis.
Conflict of interest statement. None declared.
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