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Clinical Cancer Research 14, 3416, June 1, 2008. doi: 10.1158/1078-0432.CCR-07-1824
© 2008 American Association for Cancer Research
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Imaging, Diagnosis, Prognosis

Changes in Tumor Metabolism as Readout for Mammalian Target of Rapamycin Kinase Inhibition by Rapamycin in Glioblastoma

Liu Hua Wei, Helen Su, Isabel J. Hildebrandt, Michael E. Phelps, Johannes Czernin and Wolfgang A. Weber

Authors' Affiliations: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California

Requests for reprints: Wolfgang A. Weber, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095. Phone: 310-206-2179; Fax: 310-206-4899; E-mail: wweber{at}mednet.ucla.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Purpose: Inhibition of the protein kinase mammalian target of rapamycin (mTOR) is being evaluated for treatment of a variety of malignancies. However, the effects of mTOR inhibitors are cytostatic and standard size criteria do not reliably identify responding tumors. The aim of this study was to evaluate whether response to mTOR inhibition could be assessed by positron emission tomography (PET) imaging of tumor metabolism.

Experiment Design: Glucose, thymidine, and amino acid utilization of human glioma cell lines with varying degrees of sensitivity to mTOR inhibition were assessed by measuring in vitro uptake of [18F]fluorodeoxyglucose ([18F]FDG), [18F]fluorothymidine ([18F]FLT), and [3H]L-tyrosine before and after treatment with the mTOR inhibitor rapamycin. The tumor metabolic activity in vivo was monitored by small-animal PET of tumor-bearing mice. The mechanisms underlying changes in metabolic activity were analyzed by measuring expression and functional activity of enzymes and transporters involved in the uptake of the studied imaging probes.

Results: In sensitive cell lines, rapamycin decreased [18F]FDG and [18F]FLT uptake by up to 65% within 24 hours after the start of therapy. This was associated with inhibition of hexokinase and thymidine kinase 1. In contrast, [3H]L-tyrosine uptake was unaffected by rapamycin. The effects of rapamycin on glucose and thymidine metabolism could be imaged noninvasively by PET. In sensitive tumors, [18F]FDG and [18F]FLT uptake decreased within 48 hours by 56 ± 6% and 52 ± 8%, respectively, whereas there was no change in rapamycin-resistant tumors.

Conclusions: These encouraging preclinical data warrant clinical trials evaluating [18F]FDG and [18F]FLT-PET for monitoring treatment with mTOR inhibitors in patients.

Glioblastoma multiforme is the most common malignant primary brain tumor in adults. Despite advances in diagnosis and standard therapies such as surgery, radiation, and chemotherapy, the prognosis remains poor (1, 2).

Mammalian target of rapamycin (mTOR) is a key signaling molecule involved in the growth of glioblastoma and other tumor types (2). It is a serine-threonine kinase that functions in the phosphatidylinositol 3'-kinase/Akt signaling pathway (3) by integrating extracellular signals (growth factors and hormones) with amino acid availability, and intracellular energy status to control translation rates and metabolic processes (4). In response to mitogenic stimuli, mTOR regulates the phosphorylation of p70S6 kinase, promoting translation of proteins involved in cellular proliferation, activation of angiogenesis, and metastasis formation.

Rapamycin is an antibiotic that, after binding to FKBP-12 (FK 506 binding protein), inhibits mTOR function with affinity and specificity (5). It has been used for several years as an immunosuppressive agent (6). More recently, rapamycin has been identified as a cytostatic agent with significant activity in various human cancer cell lines and tumor models (7). Based on these data, phase I, phase II, and phase III trials are being conducted for evaluation of rapamycin and its derivatives in a variety of cancers (3, 7), including glioblastomas (8, 9).

Although there is a strong rationale and preclinical evidence for the effectiveness of rapamycin as an antitumor agent, clinical trials evaluating rapamycin therapy have been limited by less than ideal techniques for assessing tumor responses to cytostatic drugs that might not, or only slowly, induce tumor shrinkage. Thus, conventional size criteria (WHO or Response Evaluation Criteria in Solid Tumors) are not readily applicable to rapamycin therapy and may underestimate the effectiveness of treatment (10). In this study, we evaluated whether positron emission tomography (PET) imaging of tumor metabolism may be used to assess tumor response to rapamycin early in the course of therapy. This approach is based on several studies that show that mTOR activity plays an important role in glucose and amino acid metabolism (11–14) and leads to cell cycle arrest in the G1 phase (15, 16). Thus, we hypothesized that blocking of mTOR signaling by rapamycin may modulate glucose and amino acid utilization as well as thymidine uptake of cancer cells. These effects of rapamycin could be imaged clinically by PET. Glucose metabolism of cancer is assessed by PET with the glucose analogue [18F]fluorodeoxyglucose ([18F]FDG). In addition, several radiolabeled amino acids and amino acid analogues are available that permit imaging of tumor amino acid transport (17). Inhibition of cellular proliferation by rapamycin could potentially be imaged with the thymidine analogue [18F]fluorothymidine ([18F]FLT; ref. 18), because accumulation of [18F]FLT has been shown to be low during the G1 phase (19). Thus, PET imaging with readily available imaging probes could provide an early readout for rapamycin activity in clinical trials. The objectives of the present study were to dissect in cell culture the effects of rapamycin on tumor cell metabolism and to use animal models to determine which imaging probe is best suited for monitoring rapamycin effects in vivo.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell lines and reagents. Rapamycin was obtained from LC Laboratories and dissolved in ethanol. Human malignant glioma cell lines U87 (with PTEN loss), LN-229 and LN-18, both with wild-type PTEN (20), were purchased from American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen), 4 mmol/L glutamine, and 100 units/mL penicillin/streptomycin at 37°C in 5% CO2.

Growth curve analysis. Cells were plated in six-well plates (Costar) at 1 x 105 per well. After 24 h, different concentrations of rapamycin (0.1-10 nmol/L) were added to each well. Viable cell numbers from triplicate wells were determined every 24 h for 3 d by trypan blue exclusion using the Vi-Cell XR instrument (Beckman Coulter).

Western blot analysis. Immunoblotting was done on 20 µg of total protein from either whole-cell lysates or membrane extracts. After SDS-PAGE, the protein was electrophoretically transferred to a polyvinylidene difluoride membrane at 100 V for 1 h at room temperature and then blocked using 5% (w/v) evaporated milk in TBS containing 0.01% (v/v) Tween 20 (TBST) for 1 h at room temperature. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies. The next day, the blots were washed in TBST buffer and incubated with goat anti-mouse or goat anti-rabbit horseradish peroxidase–conjugated secondary antibody for 1 h at room temperature. After washing in TBST buffer (three times, 10 min each wash), the immunoreactive proteins were finally visualized using ECL Plus (Amersham Biosciences).

The antibodies included anti-human phospho-p70s6, p70S6, phospho-S6 kinase, S6 kinase, PTEN, thymidine kinase 1 (TK1), and β-actin (Cell Signaling Technology). They also included anti-human GLUT-1 and GLUT-3, Na,K-ATPase (Abcam), and CD98 [human amino acid transporter heavy chain (4F2hc)].

In vitro tracer uptake studies. Synthesis and quality control of [18F]FDG and [18F]FLT were conducted by the University of California at Los Angeles Cyclotron Facility, as previously described (21). For both [18F]FDG and [18F]FLT, the radiochemical purities were >99% and the specific activities were >1,000 Ci/mmol. 3-O-[methyl-3H]glucose (3-OMeG; specific activity, 60 Ci/mmol) and [3H]L-tyrosine (specific activity 60 Ci/mmol) were obtained (Moravek Biochemicals). For all uptake studies, cells were plated in six-well plates (Costar) at 1 x 105 cells per well 1 d before the experiment. All experiments were done in triplicate and repeated at least once.

To measure [18F]FDG and [18F]FLT uptake, fresh cell culture medium containing [18F]FDG or [18F]FLT at 1 µCi/mL was added to each well. Glucose-free medium was used for [18F]FDG uptake studies. Cells were incubated with [18F]FDG or [18F]FLT at 37°C for 60 min. Cells were then rinsed twice in ice-cold PBS and then harvested with trypsin to determine cell-associated fluorine-18 radioactivity, using a Packard 5600 gamma counter (Packard).

For measurement of glucose transport, we used [3H]3-OMeG. 3-OMeG is a glucose analogue that cannot be phosphorylated by hexokinase (22); hence, the initial transport rate of 3-OMeG is a measure of glucose transport activity. Cells were incubated with [3H]3-OMeG (1 µCi/mL) and cold 3-OMeG from 10 s to 3 min at a final concentration of 0.05 mmol/L 3-OMeG, as previously described (22). Cells were then washed with ice-cold PBS supplemented with 100 mmol/L phloretin and lysed with 0.1% SDS. The cell lysates were collected; scintillation fluid (ICN) was added; and 3H radioactivity was measured in a Packard β-counter. Time-activity curves for cellular uptake of 3-OMeG were fitted by using Graph Pad Prism 4.0 (Graph Pad Software).

For measurement of [3H]L-tyrosine, uptake cells were washed thrice with Na+-free uptake medium [125 mmol/L choline-Cl, 4.8 mmol/L KCl, 1.3 mmol/L CaCl2, 1.2 mmol/L MgSO4, 25 mmol/L HEPES, 1.2 mmol/L KH2PO4, and 5.6 mmol/L glucose (pH 7.4)] and preincubated for 10 min at 37°C. The medium was then replaced by medium containing [3H]L-tyrosine at 1 µCi/mL. Following a 5-min uptake, cells were washed and radioactivity uptake was measured as described for 3-OMeG. Uptake studies were done in the presence or absence of sodium and an inhibitor of L-type amino acid transporter, 2-aminobiocyclo-(2,1,1)-heptane-2-carboxylic acid (BCH).

Uptake of all tracers was quantified as both disintegrations per minute per 106 viable (trypan blue negative) cells and as a ratio of the intracellular activity concentration and the activity concentration in the medium (cell to medium ratio) as previously described (19, 23). The cell to medium ratio describes to which extent a tracer has been concentrated within a cell and is comparable with a tumor to background ratio in a PET study. Both variables (uptake per viable cells and cell to medium ratio) showed similar changes in response to rapamycin therapy. Therefore, only the results based on the cell to medium ratio are shown in the following sections.

Cell cycle analysis. Cells were stained with propidium iodide (Calbiochem) and RNase A (Sigma) in a hypotonic buffer. The DNA content was then analyzed by a FACScan flow cytometer (Becton Dickinson) using the CellQuest 3.1 software (Becton Dickinson) for acquisition and the ModFit LT 2.0 software (Verity) for analysis. For assessment of cellular apoptosis, cells were stained with FITC-conjugated Annexin V from the Apoptosis Detection Kit II (BD Biosciences) and analyzed by a FACScan (Becton Dickinson), gating out doublets and clumps using pulse processing and collecting fluorescence above 620 nm.

Hexokinase assay. Cells were harvested, washed twice in cold PBS, and lysed in extraction buffer [45 mmol/L Tris-HCl (pH 8.2), 50 mmol/L KH2PO4, 10 mmol/L glucose, 11.1 mmol/L monothioglycerol, 0.5 mmol/L EDTA, 0.2% Triton X-100]. Whole-cell lysates were evaluated by a standard glucose-6-phosphate dehydrogenase–coupled spectrophotometric assay (24). The reaction was started by addition of extract samples and monitored at 340 nm at room temperature using a UV Spectrophotometer 8453 (Agilent Technologies).

Xenograft models. Severe combined immunodeficient (Scid/Scid) mice were purchased from The Jackson Laboratory. All animal manipulations were conducted with sterile techniques following the guidelines of the University of California at Los Angeles Animal Research Committee. U87 and LN-229 cell lines were used to generate xenografts in mice; the LN-18 cell line failed to reproducibly form tumors in SCID mice. Cells (1 x 106-5 x 106 per mouse) growing exponentially in culture were resuspended in PBS and Matrigel (BD Biosciences) and injected s.c. at the right shoulder of the animal. Mice bearing xenografts of U87 or LN-229 cells underwent either [18F]FDG- or [18F]FLT-PET/computed tomography (CT) scans when the tumor size had reached ~100 mm3 (2-3 wk after tumor cell injection). Following the PET/CT study, groups of six mice were treated once with 3 mg/kg rapamycin i.p. or vehicle as previously described (16, 25). Two days later, a follow-up PET/CT study was done using the same image acquisition variables. Following the second PET scan, tumors were excised and half of the tumor tissue was homogenized. Protein was extracted and S6 phosphorylation and TK1 levels were assessed by Western blot analysis as described above for the cell culture studies. The other half of the tumor tissue was fixed in 4% buffered formalin, processed, and embedded in paraffin. The tissue-embedded paraffin blocks were cut and slides were taken for immunohistochemical staining with an anti–Ki-67 antibody (1:100 dilution; DAKO Corp.). Tissue sections of each tumor were also stained with Harris' H&E.

To assess the effect of repeated rapamycin treatment on tumor growth, groups of six mice bearing U87 or LN-229 xenografts were treated either with vehicle or with 3 mg/kg rapamycin i.p. every other day. Tumor size was measured every other day by calipers. Tumor volume was calculated using the following equation: tumor volume = 0.5 x (greatest diameter) x (shortest diameter)2.

PET and CT imaging. MicroPET/CT scans were done using the microPET FOCUS 220 PET scanner (ref. 26; Siemens Preclinical Solutions) and MicroCAT II CT scanner (Siemens Preclinical Solutions). Mice were fasted for 12 h before [18F]FDG injection and placed on a heating pad (30°C) starting 30 min before [18F]FDG injection (27). For tracer injection and imaging, mice were anesthetized using 1.5% to 2% isoflurane. Mice were imaged in a chamber that minimizes positioning errors between PET and CT to less than 1 mm (28). Imaging was started 60 min after an i.p. injection of 200 µCi of [18F]FDG (27) or [18F]FLT via tail vein. Image acquisition time was 10 min. Images were reconstructed by filtered back-projection, using a ramp filter with a cutoff frequency of 0.5 Nyquist. Image counts per pixel per second were calibrated to activity concentrations (Bq/mL) by measuring a 3.5-cm cylinder phantom filled with a known concentration of [18F]FDG. Immediately after the PET scan, the mice underwent a 7-min microCT scan using routine image acquisition variables (70 kVp, 90 mA-s with 2-mm aluminum filters).

For display, activity concentrations were expressed as percent of the decay-corrected injected activity per gram of tissue by using the AMIDE software (29). Spherical regions of interest (2-mm diameter) were placed in the area of the tumor with the highest [18F]FDG or [18F]FLT uptake. To account for interindividual differences in [18F]FDG or [18F]FLT biodistribution, a spherical region of interest with a diameter of 2 mm was placed in the mediastinum for [18F]FLT and the liver for the [18F]FDG-PET studies as previously described (21, 30). Tumor [18F]FDG uptake was expressed as the ratio between the mean activity concentrations in the tumor region of interest divided by the mean activity concentration in the liver or mediastinum, respectively. Regions of interest were defined on fused PET/CT images generated by the AMIDE software to ensure reproducible positioning of the regions of interest.

Statistical analysis. Results are shown as mean ± 1 SE. Comparisons were made by two-tailed t tests as well as by ANOVA as appropriate. Significant associations by ANOVA were further analyzed by Bonferroni post hoc tests. P < 0.05 was considered as statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Effects of rapamycin on cell proliferation and mTOR signaling of glioblastoma cells with mutant or wide-type PTEN. In the PTEN-negative U87 cell line, rapamycin significantly inhibited cell growth at concentrations as low as 0.1 nmol/L (Fig. 1A ). In contrast, growth of the PTEN-positive LN-229 cell line was not inhibited up to a concentration of 10 nmol/L (Fig. 1B). However, the PTEN-positive LN-18 cell line showed a similar sensitivity to rapamycin as the PTEN-negative U87 cell line (Fig. 1C). Western blot analysis (Fig. 1D) confirmed the PTEN status of the tested cell lines. Phosphorylation of two downstream proteins (p70S6 kinase and S6 ribosomal protein) was abolished in U87 and LN-18 cell lines after 24 hours of incubation with rapamycin at a concentration of 0.1 nmol/L (Fig. 1D). In LN-229 cells, rapamycin showed inhibition of phosphorylation of p70S6 kinase only at the 10 nmol/L concentration. Even at this high concentration, phosphorylation of S6 was not completely abolished in this cell line (Fig. 1D).


Figure 1
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  		Fig. 1. Rapamycin inhibits cell proliferation and mTOR signaling in vitro. Cells were incubated with vehicle or with 0.1, 1, and 10 nmol/L of rapamycin for 24, 48, and 72 h, respectively. Then, the number of viable cells was determined by trypan blue staining: U87 cells (A), LN-229 cells (B), and LN-18 cells (C). D, for Western blot analysis, whole-cell proteins were prepared after incubation with rapamycin for 24 h. Twenty micrograms of protein were loaded in each lane for detecting p70S6K, S6K, PTEN, and β-actin protein levels.

 
Influence of rapamycin on [18F]FDG uptake, hexokinase activity, and glucose transporter protein expression. All untreated cell lines showed rapid [18F]FDG uptake with a cell to medium ratio of >100 after the 1-hour incubation period. As shown in Fig. 2A , rapamycin significantly inhibited [18F]FDG uptake of U87 cells within 24 hours at concentrations as low as 0.1 nmol/L. Treatment with 10 nmol/L rapamycin for 24 hours reduced uptake by 65%. In the rapamycin-resistant cell line LN-229, only 10 nmol/L of rapamycin showed a modest (30%) inhibition of [18F]FDG uptake after a 72-hour treatment (Fig. 2B). In LN-18 cells (Fig. 2C), a significant inhibition of [18F]FDG uptake was observed at rapamycin concentrations of 1 and 10 nmol/L.


Figure 2
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  		Fig. 2. Influence of rapamycin on cellular glucose utilization. A to C, [18F]FDG uptake of the three studied cell lines treated with rapamycin at the indicated concentrations or vehicle. D, effect of rapamycin on the expression of Glut1 and Glut3. U87 cells were incubated with vehicle or 10 nmol/L of rapamycin for 24 h. Then, the expression of Glut1 and Glut3 was determined by Western blots of whole-cell lysates and cell membrane fractions. Fraction purity was confirmed by performing Western blots for Na+,K+-ATPase (cell membrane marker). E, effect of rapamycin on 3-OMeG uptake. U87 cells were incubated with vehicle or 10 nmol/L of rapamycin for 24 h. Then, 3-OMeG uptake was measured for 3 min. F, effect of rapamycin on hexokinase activity. U87 cells were incubated with vehicle or with 0.1, 1, and 10 nmol/L of rapamycin for 24 h. Whole-cell extracts were prepared to assess the hexokinase activity. *, P < 0.05; **, P < 0.01 compared with vehicle control.

 
Next, we determined if the inhibition of [18F]FDG uptake by rapamycin was attributed to the inhibition of glucose transporter expression or translocation of transporters from the cell membrane to the cytoplasm. Figure 2D illustrates that in U87 cells, total and cell membrane–associated glucose transporter protein levels were not affected by treatment with 10 nmol/L rapamycin for 24 hours. Furthermore, the uptake rate of [3H]3-OMeG, a marker for glucose transport, was not reduced by rapamycin therapy (Fig. 2E).

These findings indicated that the observed decrease in [18F]FDG uptake could not be explained by a reduction of glucose transport activity. However, hexokinase activity was significantly reduced by rapamycin therapy. Basal hexokinase activity in U87 cells was 54 ± 6 milliunits/mg protein. Treatment with rapamycin for 24 hours resulted in a statistically significant (P < 0.01) dose-dependent decrease of hexokinase activity as shown in Fig. 2F.

Influence of rapamycin on [18F]FLT uptake, TK1 expression, cell cycle progression, and apoptosis. Cellular uptake of [18F]FLT was determined after incubating for 24, 48, and 72 hours in the presence of rapamycin or vehicle. In untreated cells, the [18F]FLT cell to medium ratio was >100 for all cell lines. As shown in Fig. 3A and B , rapamycin at 1 and 10 nmol/L significantly diminished [18F]FLT uptakes in both U87 and LN-18 by up to 60% (P < 0.01). No significant decrease in [18F]FLT uptake was observed in LN-229 cells (Fig. 3C). Because TK1 activity is considered to be the most important determinant of [18F]FLT uptake (19), we investigated the effect of rapamycin on TK1 expression by Western blot analysis. As shown in Fig. 3D, rapamycin attenuated TK1 protein expression in U87 and LN-18, but not in LN-229 cell lines. In LN-229 cells, rapamycin actually induced a moderate increase in TK1 expression levels. This was not paralleled by an increased growth rate (Fig. 1B) or FLT uptake (Fig. 3C), suggesting that TK1 activity is controlled by posttranslational mechanisms as previously described (31). We next determined whether treatment of U87 cells with rapamycin leads to an accumulation of the cells in the G0-G1 phase. As shown in Fig. 3E, the percentage of G0-G1 phase cells increased in rapamycin-treated U87 cells, indicating that rapamycin arrests cells at the G1 checkpoint. Finally, we explored whether rapamycin caused apoptosis in U87 cells as determined by the binding of Annexin V. The percentage of apoptotic cells was 2.3% in untreated U87 cells and 2.9% with rapamycin-treated cells (10 nmol/L for 24 h; Fig. 3F). Thus, rapamycin did not induce apoptosis in U87 cells even at the highest tested concentration.


Figure 3
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  		Fig. 3. Influence of rapamycin on [18F]FLT uptake, TK1 expression, cell cycle distribution, and apoptosis. A to C, [18F]FLT uptake of the three studied cell lines treated with rapamycin at the indicated concentrations or vehicle. D, effect of rapamycin on TK1 expression. U87, LN-18, and LN-229 cells were incubated with the indicated concentrations for 24 h. E, cell cycle distribution of rapamycin-treated cells and controls. U87 cells were incubated with the indicated concentrations of rapamycin for 24 h. Cells were then stained with propidium iodide and subjected to FACS. F, Annexin V staining of U87 cells 24 h after treatment with 10 nmol/L rapamycin or vehicle. *, P < 0.05; **, P < 0.01 compared with vehicle control.

 
Influence of rapamycin on the L-type amino acid transport system. In untreated cells, the cell to medium ratio was >10 after the 5-minute uptake period. As shown in Fig. 4A , treatment with rapamycin for 24 hours at 0.1, 1, and 10 nmol rapamycin did not affect [3H]L-tyrosine uptake in U87 cells. BCH, a competitive inhibitor of the amino acid transport system L completely blocked the [3H]L-tyrosine uptake of U87 cells, whereas sodium depletion had no significant effect on [3H]L-tyrosine uptake (data not shown). Because these findings indicated that the amino acid transport system L mediates uptake of tyrosine in U87 cells, we did FACS analysis to determine the cell surface expression of 4F2hc (CD98), the heavy chain of the L-type transporters LAT1 and LAT2 (Fig. 4B and C). These studies showed that U87 cells express 4F2hc but there was no inhibition of 4F2hc expression following rapamycin treatment (Fig. 4C).


Figure 4
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  		Fig. 4. Effects of rapamycin on the L-type amino acid transport system. A, uptake of [3H]tyrosine. U87 cells were incubated with vehicle or with 0.1, 1, and 10 nmol/L of rapamycin for 24 h and then incubated with 1 µCi/mL [3H]L-tyrosine for 5 min. B and C, cell surface expression of CD98. U87 cells were incubated with vehicle (B) or 10 nmol/L rapamycin (C) for 24 h; cells were then washed and incubated with an anti-CD98 primary antibody and then with a FITC-conjugated second antibody. Y-axis is side scatter (SSC). FACS analysis was then done to determine CD98 protein levels on the cell surface.

 
Rapamycin inhibits [18F]FDG and [18F]FLT uptake in vivo. In U87 tumors treated with rapamycin, [18F]FDG uptake was reduced to 44 ± 6% of the baseline value (P < 0.01) as shown in Fig. 5A and C . Liver activity did not change with rapamycin therapy (83.2 ± 6.4 counts/pixel/s at baseline and 84 ± 5.4 counts/pixel/s at follow up); thus, the decrease of the tumor to liver ratio was not due to an increase in liver activity. In contrast to U87 tumors, [18F]FDG uptake did not significantly change in LN-229 tumors (P = 0.3; Fig. 5B and D). After treatment with rapamycin, fasting serum glucose levels significantly increased in both xenograft models (from 80 ± 7 to 141 ± 11 mg/mL in animals bearing U87 tumors and from 70 ± 9 to 144 ± 16 mg/mL in animals bearing LN-229 tumors, P < 0.05 for both cell lines). U87 and LN-229 xenografts, which received vehicle treatment, showed no significant changes of [18F]FDG uptake from the baseline to the follow-up scan (P > 0.05 for both cell lines). When comparing FDG uptake of rapamycin-treated tumors with that of vehicle-treated tumors, there was a highly significant difference for U87 tumors (2.26 ± 0.22 versus 5.1 ± 0.4, P = 0.001). In contrast, there was no significant difference for vehicle- and rapamycin-treated LN-229 xenografts (4.9 ± 0.6 versus 4.1 ± 0.4, P = 0.27).


Figure 5
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  		Fig. 5. [18F]FDG- and [18F]FLT-PET/CT scans of tumor xenografts before and after treatment with rapamycin. U87 and LN-229 xenografts received either [18F]FDG-PET/CT or [18F]FLT-PET/CT scans before and after one dose of rapamycin (3 mg/kg), 48 h after the baseline scan. Axial [18F]FDG-PET/CT scans of representative tumor are shown in A and B. Changes in tumor [18F]FDG uptake are summarized in C and D. E and F show [18F]FLT-PET scans of U87 and LN-229 xenografts before and after one dose of rapamycin. Changes in tumor [18F]FLT uptake are summarized in F and H. **, P < 0.01; *, P < 0.05 for comparison of pretreatment and posttreatment scans.

 
Treatment with rapamycin significantly reduced [18F]FLT uptake of U87 tumors to 48 ± 7% of baseline level (Fig. 5E and G; P < 0.01). Mediastinal blood pool activity did not change with rapamycin therapy (70 ± 1.2 counts/pixel/s before therapy and 71 ± 3.2 counts/pixel/s after therapy). In LN-229 tumors (Fig. 5F and H), no significant changes in [18F]FLT uptake were observed after rapamycin therapy (P = 0.2). When comparing FLT uptake of the rapamycin-treated tumors with that of the vehicle-treated tumors, there was a significant difference for U87 tumors (0.8 ± 0.1 versus 2.1 ± 0.2, P = 0.01). In contrast, there was no significant difference for vehicle- and rapamycin-treated LN-229 xenografts (4.9 ± 0.5 versus 5.1 ± 0.1, P = 0.18). Tumor size also did not significantly change at the time of the follow-up PET scan (P > 0.3 for both LN-229 and U87 tumors). However, continued treatment with rapamycin efficiently inhibited tumor growth in U87, but not in LN-229, xenografts (Fig. 6 ).


Figure 6
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  		Fig. 6. Effect of rapamycin therapy on mTOR activity and tumor cell proliferation in vivo. A, Western blot analyses of tumor tissue collected after the follow-up PET scan (48 h after i.p. injection of 3 mg/kg rapamycin). B and C, H&E and Ki67 staining of U87 and LN-229 tumors. Tumors were excised 48 h after injection of 3 mg/kg rapamycin or vehicle. D and E, tumor growth curves of U87 and LN-229 tumors treated with 3 mg/kg rapamycin i.p. or vehicle every other day for 2 wk.

 
Reduction of tumor [18F]FDG and [18F]FLT uptake correlates with rapamycin-induced growth inhibition in vivo. Western blot analysis of tumor tissue excised after the follow-up PET scans showed that rapamycin therapy had abolished S6 protein phosphorylation in both U87 and LN-229 xenografts (Fig. 6A). However, TK1 levels were inhibited only in U87 tumors. Histologic analysis provided no evidence for treatment-induced necrosis in U87 and LN-229 tumors (Fig. 6B and C). However, Ki-67 staining was markedly reduced in U87 tumors (Fig. 6B) but not in LN-229 xenografts (Fig. 6C). When mice were treated with 3 mg/kg rapamycin every other day for 2 weeks, the volume of U87 xenografts slowly declined. Vehicle-treated U87 tumors showed a rapid exponential growth (Fig. 6D). In contrast, treatment with rapamycin did not inhibit growth of LN-229 xenografts (Fig. 6E).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
This study shows that mTOR inhibition by rapamycin therapy can cause a rapid reduction of glucose and thymidine use of glioblastoma cell lines. These metabolic changes correlated well with rapamycin-induced growth inhibition in vitro and in vivo. If confirmed in clinical trials, these findings would suggest that PET imaging with FDG or FLT may be used to monitor treatment with rapamycin and other mTOR inhibitors in patients. In contrast, L-type amino acid transport, which is considered to mediate the uptake of most clinically available amino acid probes (17, 32), does not seem to reflect mTOR inhibition in glioblastomas. Therefore, PET imaging with [18F]FDG or [18F]FLT may be preferable to radiolabeled amino acids for monitoring treatment responses to rapamycin.

The rationale for monitoring tumor glucose and amino acid metabolism for assessing the effects of mTOR inhibitors is based on the well-established role of mTOR in cellular metabolism and growth factor signaling. Signaling by mTOR is activated through Akt-induced inhibition of the tuberous sclerosis complex TSC1/TSC2, which inhibits mTOR activity (33). In various tumor models, activation of Akt or HIF-1 {alpha} has been shown to lead to increased expression of glycolytic enzymes and transporters (34, 35). These effects of AKT and HIF-1{alpha} were blocked by rapamycin, indicating that mTOR plays a role in regulating glucose use of cancer cells.

Transport of glucose across the plasma membrane of mammalian cells is the first step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Overexpression of the GLUT1 and GLUT3 subtypes has been described in many cancers (36). Hexokinase, which converts glucose to glucose-6-phosphate, helps maintain the glucose concentration gradient that results in the movement of glucose into cells (37). The glucose analogue [18F]FDG traces these first two steps of glucose metabolism but cannot be metabolized further. Our results indicate that [18F]FDG uptake and hexokinase activity are rapidly decreased in cells that are sensitive to rapamycin, whereas there is little effect on [18F]FDG uptake of rapamycin in resistant cells. These metabolic effects of rapamycin could be imaged noninvasively by microPET within 48 hours after a single dose of rapamycin, suggesting that [18F]FDG-PET may potentially be used to monitor rapamycin therapy in the clinic.

We found, however, that rapamycin therapy causes hyperglycemia in mice. Because [18F]FDG competes with glucose for transport and phosphorylation, hyperglycemia can reduce tumor [18F]FDG uptake. Hyperglycemia has also been reported in clinical studies of rapamycin analogues (10, 38). However, hyperglycemia cannot explain the observed reduction in [18F]FDG uptake in U87 tumors because [18F]FDG uptake of rapamycin-resistant LN-229 tumors was unchanged following rapamycin therapy. Furthermore, our in vitro studies show that rapamycin modulates glucose metabolic activity of cancer cells.

Rapamycin has cytostatic effects in a variety of preclinical tumor models. This effect is caused by a decrease of cyclin D1 expression and an increase in p27, leading to an arrest of cells in G1 (7). The thymidine analogue FLT is a substrate of TK1, whose expression levels and functional activity are low during the G1 phase of the cell cycle (31). TK1 activity is the key determinant for [18F]FLT uptake by cancer cells (19). The uptake of [18F]FLT also correlates with immunohistochemical proliferation markers such as Ki67 in glioblastoma (39). Based on this evidence, we tested the ability of [18F]FLT-PET to monitor the cytostatic effects of rapamycin in glioblastoma. Inhibition of cellular proliferation by rapamycin caused a rapid decrease in [18F]FLT uptake, which was paralleled by a decrease in TK1 protein expression and accumulation of cells in the G1 phase. These changes in [18F]FLT uptake were also evident in vivo by microPET imaging and were allowed to differentiate between rapamycin-sensitive and rapamycin-insensitive tumors within 48 hours after start of therapy (Fig. 6).

PET/CT imaging of mice is associated with a considerably higher radiation exposure than PET/CT in humans. Our CT protocol resulted in a tumor dose of ~45 mGy (40) and the injection of 200 µCi [18F]FDG or [18F]FLT caused a tumor dose of ~200 to 400 mGy (41). Although this radiation dose is unlikely to have a significant effect on tumor growth, one cannot rule out that radiation exposure combined with the effects of fasting and anesthesia could have some effect on tumor growth, metabolism, or tracer biodistribution. To ensure that the observed changes in [18F]FDG or [18F]FLT uptake were not confounded by these factors, we also imaged vehicle-treated animals. In mice bearing U87 tumors, one dose of rapamycin significantly reduced FDG and FLT uptake compared with the vehicle-treated animals. In contrast, there were no significant differences between rapamycin- and vehicle-treated LN-229 tumors. In addition, we generated growth curves in mice that were not imaged by PET/CT. These growth curves showed a marked therapeutic effect of rapamycin on U87 xenografts, but not on LN-229 xenografts (Fig. 6).

The observed decrease in [18F]FDG or [18F]FLT uptake was not explained by cell death because there was no decrease in tumor size in vivo and no increase in the number of trypan blue or Annexin V–positive cells in vitro. These results are consistent with previous studies that have found that a cytotoxic response to rapamycin is infrequent (7). In clinical trials, tumor shrinkage in response to mTOR inhibition has been rare, even when patient survival was significantly improved (38).

Glioblastoma exhibit markedly increased uptake of neutral amino acids, which is believed to result from an increased expression and activity of the LAT-1 transporter (42, 43). The LAT-1 transport is one of the transporters of the L transport system, which transports large neutral amino acids such as tyrosine. The LAT-1 transporter is composed of two covalently linked protein chains called 4F2 light chain and 4F2 heavy chain (4F2hc) or CD98 (44). Studies have indicated that in growth-factor–deprived leukemia cells, rapamycin can inhibit Akt-stimulated amino acid uptake (45). In this context, rapamycin caused a translocation of 4F2hc from the plasma membrane to the cytosol. In contrast, in the presence of growth factors, inhibition of mTOR with rapamycin did not cause translocation of 4F2hc (46). In this study, we found that U87 cells expressed a high level of LAT1/4F2hc and that tyrosine uptake is mediated by the L system. However, treatment with rapamycin did not affect L-tyrosine uptake by U87 cell line despite significant growth inhibition. Thus, our data suggest that L-tyrosine and its analogues are less suited than [18F]FDG and [18F]FLT for monitoring rapamycin therapy in the clinic.

As models for rapamycin-sensitive and rapamycin-insensitive tumors, we used cell lines with and without deletion of the tumor-suppressor gene PTEN, because previous studies had indicated that loss of PTEN is associated with increased sensitivity to rapamycin (15, 47). In accordance with these previous observations, we found that growth of the PTEN null U87 cell lines was inhibited at low concentrations of rapamycin (0.1 nmol/L), whereas 100-fold higher concentrations had no growth-inhibitory effect on PTEN wild-type LN-229 cells. However, LN-18 cells, which also express wild-type PTEN (20), showed a similar sensitivity to rapamycin as U87 cells in vitro, indicating that PTEN status does not completely reflect cell vulnerability to mTOR inhibition. Similar observations have been made by others in glioblastoma (48) and other tumor types (47). The lack of reliable molecular markers to predict tumor responsiveness to rapamycin emphasizes the need for techniques to monitor tumor response early in the course of therapy. Furthermore, rapamycin showed a concentration-dependent inhibition of the phosphorylation of the mTOR targets 70S6K and ribosomal protein S6 proteins in PTEN wild-type LN-229 cells, although no inhibition of cell growth was observed in this cell line. Similarly, one dose of rapamycin abolished S6 phosphorylation in vivo, but did not inhibit growth of LN-229 xenografts. This suggests that phosphorylation of p70S6K and S6 are not essential in regulating the growth of LN-229 cells. This confirms previous observations (48) that inhibition of phosphorylation of P70S6K and S6 are not sufficient for a growth inhibitory effect of rapamycin. Thus, phosphorylation of S6, which has been used to monitor rapamycin therapy in clinical trials, is a marker of target inhibition, but not necessarily of tumor response (49, 50).

In conclusion, the results of the present study suggest that imaging of tumor glucose and thymidine utilization may be useful for monitoring the growth-inhibitory effects of rapamycin. If validated in clinical trials, PET imaging with [18F]FDG or [18F]FLT could complement pretherapeutic analysis of the PTEN status and assessment of changes in S6 phosphorylation. In contrast, imaging of L-type amino acid transport with tyrosine analogues is unlikely to assess tumor response early in the course of therapy. This study also illustrates how in vitro studies and small-animal PET imaging can be used to select molecular imaging probes for further evaluation in clinical trials.


   Disclosure of Potential Conflicts of Interest
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 Results
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References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
We thank Waldemar Ladno, Antonia Luu, Judy Edwards, and Dr. David Stout for excellent technical assistance with animal imaging.


   Footnotes
 
Grant support: University of California at Los Angeles Center for In vivo Imaging in Cancer Biology (National Cancer Institute P50 CA086306) and the University of California at Los Angeles Interactive Multimedia Education at a Distance (DE-FC03-87E60615) grants, the UCLA 1CM1C (NC1 P50 CA086306), and the UCLA IMED CD1-7-C03-87E60615 grants.

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.

Received 7/24/07; revised 12/11/07; accepted 1/18/08.


   References
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 Abstract
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 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Mitchell P, Ellison DW, Mendelow AD. Surgery for malignant gliomas: mechanistic reasoning and slippery statistics. Lancet Neurol 2005;4:413–22.[CrossRef][Medline]
  2. Maher EA, Furnari FB, Bachoo RM, et al. Malignant glioma: genetics and biology of a grave matter. Genes Dev 2001;15:1311–33.[Free Full Text]
  3. Abraham RT, Gibbons JJ. The mammalian target of rapamycin signaling pathway: twists and turns in the road to cancer therapy. Clin Cancer Res 2007;13:3109–14.[Abstract/Free Full Text]
  4. Gingras AC, Gygi SP, Raught B, et al. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev 1999;13:1422–37.[Abstract/Free Full Text]
  5. Huang S, Houghton PJ. Inhibitors of mammalian target of rapamycin as novel antitumor agents: from bench to clinic. Curr Opin Investig Drugs 2002;3:295–304.[Medline]
  6. Yatscoff RW, LeGatt DF, Kneteman NM. Therapeutic monitoring of rapamycin: a new immunosuppressive drug. Ther Drug Monit 1993;15:478–82.[Medline]
  7. Faivre S, Kroemer G, Raymond E. Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 2006;5:671–88.[CrossRef][Medline]
  8. Doherty L, Gigas DC, Kesari S, et al. Pilot study of the combination of EGFR and mTOR inhibitors in recurrent malignant gliomas. Neurology 2006;67:156–8.[Abstract/Free Full Text]
  9. Reardon DA, Quinn JA, Vredenburgh JJ, et al. Phase 1 trial of gefitinib plus sirolimus in adults with recurrent malignant glioma. Clin Cancer Res 2006;12:860–8.[Abstract/Free Full Text]
  10. Galanis E, Buckner JC, Maurer MJ, et al. Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group Study. J Clin Oncol 2005;23:5294–304.[Abstract/Free Full Text]
  11. Sharma S, Guthrie PH, Chan SS, Haq S, Taegtmeyer H. Glucose phosphorylation is required for insulin-dependent mTOR signalling in the heart. Cardiovasc Res 2007.
  12. Fuchs BC, Finger R, Onan MC, Bode BP. ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells. Am J Physiol Cell Physiol 2007.
  13. Kimball SR, Jefferson LS. Signaling pathways and molecular mechanisms through which branched-chain amino acids mediate translational control of protein synthesis. J Nutr 2006;136:227–31S.[CrossRef]
  14. Raught B, Gingras AC, Sonenberg N. The target of rapamycin (TOR) proteins. Proc Natl Acad Sci U S A 2001;98:7037–44.[Abstract/Free Full Text]
  15. Vignot S, Faivre S, Aguirre D, Raymond E. mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol 2005;16:525–37.[Abstract/Free Full Text]
  16. Boffa DJ, Luan F, Thomas D, et al. Rapamycin inhibits the growth and metastatic progression of non-small cell lung cancer. Clin Cancer Res 2004;10:293–300.[Abstract/Free Full Text]
  17. Herholz K, Coope D, Jackson A. Metabolic and molecular imaging in neuro-oncology. Lancet Neurol 2007;6:711–24.[CrossRef][Medline]
  18. Shields AF, Grierson JR, Dohmen BM, et al. Imaging proliferation in vivo with [F-18]FLT and positron emission tomography. Nat Med 1998;4:1334–6.[CrossRef][Medline]
  19. Schwartz JL, Tamura Y, Jordan R, Grierson JR, Krohn KA. Monitoring tumor cell proliferation by targeting DNA synthetic processes with thymidine and thymidine analogs. J Nucl Med 2003;44:2027–32.[Abstract/Free Full Text]
  20. Ishii N, Maier D, Merlo A, et al. Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines. Brain Pathol 1999;9:469–79.[Medline]
  21. Su H, Bodenstein C, Dumont RA, et al. Monitoring tumor glucose utilization by positron emission tomography for the prediction of treatment response to epidermal growth factor receptor kinase inhibitors. Clin Cancer Res 2006;12:5659–67.[Abstract/Free Full Text]
  22. Aloj L, Caraco C, Jagoda E, Eckelman WC, Neumann RD. Glut-1 and hexokinase expression: relationship with 2-fluoro-2-deoxy-D-glucose uptake in A431 and T47D cells in culture. Cancer Res 1999;59:4709–14.[Abstract/Free Full Text]
  23. Sasajima T, Miyagawa T, Oku T, Gelovani JG, Finn R, Blasberg R. Proliferation-dependent changes in amino acid transport and glucose metabolism in glioma cell lines. Eur J Nucl Med Mol Imaging 2004;31:1244–56.[Medline]
  24. Robey RB, Raval BJ, Ma J, Santos AV. Thrombin is a novel regulator of hexokinase activity in mesangial cells. Kidney Int 2000;57:2308–18.[CrossRef][Medline]
  25. Hale DA, Gottschalk R, Fukuzaki T, Wood ML, Maki T, Monaco AP. Superiority of sirolimus (rapamycin) over cyclosporine in augmenting allograft and xenograft survival in mice treated with antilymphocyte serum and donor-specific bone marrow. Transplantation 1997;63:359–64.[CrossRef][Medline]
  26. Tai YC, Ruangma A, Rowland D, et al. Performance evaluation of the microPET focus: a third-generation microPET scanner dedicated to animal imaging. J Nucl Med 2005;46:455–63.[Abstract/Free Full Text]
  27. Fueger BJ, Czernin J, Hildebrandt I, et al. Impact of animal handling on the results of 18F-FDG PET studies in mice. J Nucl Med 2006;47:999–1006.[Abstract/Free Full Text]
  28. Chow P, Stout D, Komisopoulou E, Chatziioannou A. A method of image registration for small animal, multi-modality imaging. Phys Med Bil 2006;51:379–90.[CrossRef]
  29. Loening AM, Gambhir SS. AMIDE: a free software tool for multimodality medical image analysis. Mol Imaging 2003;2:131–7.[CrossRef][Medline]
  30. Waldherr C, Mellinghoff IK, Tran C, et al. Monitoring antiproliferative responses to kinase inhibitor therapy in mice with 3'-deoxy-3'-18F-fluorothymidine PET. J Nucl Med 2005;46:114–20.[Abstract/Free Full Text]
  31. Munch-Petersen B, Cloos L, Jensen HK, Tyrsted G. Human thymidine kinase 1. Regulation in normal and malignant cells. Adv Enzyme Regul 1995;35:69–89.[CrossRef][Medline]
  32. Lahoutte T, Caveliers V, Camargo SM, et al. SPECT and PET amino acid tracer influx via system L (h4F2hc-hLAT1) and its transstimulation. J Nucl Med 2004;45:1591–6.[Abstract/Free Full Text]
  33. Bhaskar PT, Hay N. The two TORCs and Akt. Dev Cell 2007;12:487–502.[CrossRef][Medline]
  34. Majumder PK, Febbo PG, Bikoff R, et al. mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat Med 2004;10:594–601.[CrossRef][Medline]
  35. Thomas GV, Tran C, Mellinghoff IK, et al. Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med 2006;12:122–7.[CrossRef][Medline]
  36. Macheda ML, Rogers S, Best JD. Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer. J Cell Physiol 2005;202:654–62.[CrossRef][Medline]
  37. Kim JW, Dang CV. Multifaceted roles of glycolytic enzymes. Trends Biochem Sci 2005;30:142–50.[CrossRef][Medline]
  38. Hudes G, Carducci M, Tomczak P, et al. Temsirolimus, interferon {alpha}, or both for advanced renal-cell carcinoma. N Engl J Med 2007;356:2271–81.[Abstract/Free Full Text]
  39. Chen W, Cloughesy T, Kamdar N, et al. Imaging proliferation in brain tumors with 18F-FLT PET: comparison with 18F-FDG. J Nucl Med 2005;46:945–52.[Abstract/Free Full Text]
  40. Taschereau R, Chow PL, Chatziioannou AF. Monte Carlo simulations of dose from microCT imaging procedures in a realistic mouse phantom. Med Phys 2006;33:216–24.[CrossRef][Medline]
  41. Taschereau R, Chatziioannou AF. Monte Carlo simulations of absorbed dose in a mouse phantom from 18-fluorine compounds. Med Phys 2007;34:1026–36.[CrossRef][Medline]
  42. Kanai Y, Segawa H, Miyamoto K, Uchino H, Takeda E, Endou H. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J Biol Chem 1998;273:23629–32.[Abstract/Free Full Text]
  43. Sang J, Lim YP, Panzica M, Finch P, Thompson NL. TA1, a highly conserved oncofetal complementary DNA from rat hepatoma, encodes an integral membrane protein associated with liver development, carcinogenesis, and cell activation. Cancer Res 1995;55:1152–9.[Abstract/Free Full Text]
  44. Fuchs BC, Bode BP. Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime? Semin Cancer Biol 2005;15:254–66.[CrossRef][Medline]
  45. Edinger AL, Thompson CB. Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake. Mol Biol Cell 2002;13:2276–88.[Abstract/Free Full Text]
  46. Edinger AL, Linardic CM, Chiang GG, Thompson CB, Abraham RT. Differential effects of rapamycin on mammalian target of rapamycin signaling functions in mammalian cells. Cancer Res 2003;63:8451–60.[Abstract/Free Full Text]
  47. Neshat MS, Mellinghoff IK, Tran C, et al. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci U S A 2001;98:10314–9.[Abstract/Free Full Text]
  48. Hosoi H, Dilling MB, Liu LN, et al. Studies on the mechanism of resistance to rapamycin in human cancer cells. Mol Pharmacol 1998;54:815–24.[Abstract/Free Full Text]
  49. Duran I, Kortmansky J, Singh D, et al. A phase II clinical and pharmacodynamic study of temsirolimus in advanced neuroendocrine carcinomas. Br J Cancer 2006;95:1148–54.[CrossRef][Medline]
  50. Peralba JM, DeGraffenried L, Friedrichs W, et al. Pharmacodynamic evaluation of CCI-779, an inhibitor of mTOR, in cancer patients. Clin Cancer Res 2003;9:2887–92.[Abstract/Free Full Text]



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