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Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

Tardito S, Oudin A, Ahmed SU, Fack F, Keunen O, Zheng L, Miletic H, Sakariassen PØ, Weinstock A, Wagner A, Lindsay SL, Hock AK, Barnett SC, Ruppin E, Mørkve SH, Lund-Johansen M, Chalmers AJ, Bjerkvig R, Niclou SP, Gottlieb E - Nat. Cell Biol. (2015)

Bottom Line: However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation.Moreover, Gln-starved cells are not rescued by TCA cycle replenishment.In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons.

View Article: PubMed Central - PubMed

Affiliation: Cancer Metabolism Research Unit, Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

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Gln metabolism in differentiated (DIFF) and GBM Stem-like (GSC) primary human GBM cells. (a) Protein expression was assessed in E2, R10 and R24 cells maintained in DMEM/F-12 and supplemented as described in the Methods section. Arrow points to SOX2 specific band. A representative experiment repeated twice is shown. Unprocessed scans of western blots are shown in Supplementary Figure 8. (b) Cells were incubated in SMEM supplemented as described in the Methods section, +/− 0.65mM Gln and 1mM MSO as indicated. Mean ± S.E.M. n=3 independent experiments. (c-d) The exchange rates of Gln (c) and Glu (d) isotopologues in cells incubated for 24h in SMEM +/− 0.65mM Gln supplemented with of 0.8mM . Mean ± S.E.M. n=3 independent experiments. (e-h) The Intracellular content of Gln (e), Glu (f), citrate (g), and AMP (h) isotopologues in cells incubated as in (c-d). Mean ± S.E.M. n=3 independent experiments.
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Figure 5: Gln metabolism in differentiated (DIFF) and GBM Stem-like (GSC) primary human GBM cells. (a) Protein expression was assessed in E2, R10 and R24 cells maintained in DMEM/F-12 and supplemented as described in the Methods section. Arrow points to SOX2 specific band. A representative experiment repeated twice is shown. Unprocessed scans of western blots are shown in Supplementary Figure 8. (b) Cells were incubated in SMEM supplemented as described in the Methods section, +/− 0.65mM Gln and 1mM MSO as indicated. Mean ± S.E.M. n=3 independent experiments. (c-d) The exchange rates of Gln (c) and Glu (d) isotopologues in cells incubated for 24h in SMEM +/− 0.65mM Gln supplemented with of 0.8mM . Mean ± S.E.M. n=3 independent experiments. (e-h) The Intracellular content of Gln (e), Glu (f), citrate (g), and AMP (h) isotopologues in cells incubated as in (c-d). Mean ± S.E.M. n=3 independent experiments.

Mentions: To corroborate the causal link between Gln biosynthesis and Gln-dependency, GS was overexpressed in LN18 cells, which display low GS levels and high sensitivity to Gln deprivation. To this end, LN18-derived clones stably expressing infra-Red Fluorescent Protein only (iRFP)26,27 or iRFP and GS were established. To eliminate intrinsic clonal variability, GS expression and its effect on growth under Gln starvation were evaluated in multiple clones (6 iRFP and 9 iRFP-GS). After five days of starvation, growth of iRFP control cells reached, on average, 16% ± 5 % of control Gln-fed cells, while iRFP-GS clones reached on average, 54% ± 12 % (Fig. 4a). Under Gln-supplementation, the iRFP-GS5 clone proliferated slower than iRFP controls. This was not rectified by GS inhibition with MSO (Fig. 5b), consistent with a reported non-metabolic, anti-proliferative role of GS28. Nevertheless, iRFP-GS5, but not control iRFP4 cells, proliferated and formed colonies in Gln-free medium and this growth advantage was blocked by MSO (Fig. 4b-c). These results imply that under Gln starvation the amidation of Glu via GS sustains cell growth. In line with this, when supplemented with 15N1-ammonia, GS-expressing cells displayed 15N incorporation into ~50% of the total Gln pool, even when Gln fed (Fig 4d). When incubated with 15N1-ammonia without Gln, residual intracellular Gln was higher in iRFP-GS5 cells compared to control iRFP4 cells, and produced entirely by GS as judged by 15N incorporation (Fig. 4d).


Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

Tardito S, Oudin A, Ahmed SU, Fack F, Keunen O, Zheng L, Miletic H, Sakariassen PØ, Weinstock A, Wagner A, Lindsay SL, Hock AK, Barnett SC, Ruppin E, Mørkve SH, Lund-Johansen M, Chalmers AJ, Bjerkvig R, Niclou SP, Gottlieb E - Nat. Cell Biol. (2015)

Gln metabolism in differentiated (DIFF) and GBM Stem-like (GSC) primary human GBM cells. (a) Protein expression was assessed in E2, R10 and R24 cells maintained in DMEM/F-12 and supplemented as described in the Methods section. Arrow points to SOX2 specific band. A representative experiment repeated twice is shown. Unprocessed scans of western blots are shown in Supplementary Figure 8. (b) Cells were incubated in SMEM supplemented as described in the Methods section, +/− 0.65mM Gln and 1mM MSO as indicated. Mean ± S.E.M. n=3 independent experiments. (c-d) The exchange rates of Gln (c) and Glu (d) isotopologues in cells incubated for 24h in SMEM +/− 0.65mM Gln supplemented with of 0.8mM . Mean ± S.E.M. n=3 independent experiments. (e-h) The Intracellular content of Gln (e), Glu (f), citrate (g), and AMP (h) isotopologues in cells incubated as in (c-d). Mean ± S.E.M. n=3 independent experiments.
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Figure 5: Gln metabolism in differentiated (DIFF) and GBM Stem-like (GSC) primary human GBM cells. (a) Protein expression was assessed in E2, R10 and R24 cells maintained in DMEM/F-12 and supplemented as described in the Methods section. Arrow points to SOX2 specific band. A representative experiment repeated twice is shown. Unprocessed scans of western blots are shown in Supplementary Figure 8. (b) Cells were incubated in SMEM supplemented as described in the Methods section, +/− 0.65mM Gln and 1mM MSO as indicated. Mean ± S.E.M. n=3 independent experiments. (c-d) The exchange rates of Gln (c) and Glu (d) isotopologues in cells incubated for 24h in SMEM +/− 0.65mM Gln supplemented with of 0.8mM . Mean ± S.E.M. n=3 independent experiments. (e-h) The Intracellular content of Gln (e), Glu (f), citrate (g), and AMP (h) isotopologues in cells incubated as in (c-d). Mean ± S.E.M. n=3 independent experiments.
Mentions: To corroborate the causal link between Gln biosynthesis and Gln-dependency, GS was overexpressed in LN18 cells, which display low GS levels and high sensitivity to Gln deprivation. To this end, LN18-derived clones stably expressing infra-Red Fluorescent Protein only (iRFP)26,27 or iRFP and GS were established. To eliminate intrinsic clonal variability, GS expression and its effect on growth under Gln starvation were evaluated in multiple clones (6 iRFP and 9 iRFP-GS). After five days of starvation, growth of iRFP control cells reached, on average, 16% ± 5 % of control Gln-fed cells, while iRFP-GS clones reached on average, 54% ± 12 % (Fig. 4a). Under Gln-supplementation, the iRFP-GS5 clone proliferated slower than iRFP controls. This was not rectified by GS inhibition with MSO (Fig. 5b), consistent with a reported non-metabolic, anti-proliferative role of GS28. Nevertheless, iRFP-GS5, but not control iRFP4 cells, proliferated and formed colonies in Gln-free medium and this growth advantage was blocked by MSO (Fig. 4b-c). These results imply that under Gln starvation the amidation of Glu via GS sustains cell growth. In line with this, when supplemented with 15N1-ammonia, GS-expressing cells displayed 15N incorporation into ~50% of the total Gln pool, even when Gln fed (Fig 4d). When incubated with 15N1-ammonia without Gln, residual intracellular Gln was higher in iRFP-GS5 cells compared to control iRFP4 cells, and produced entirely by GS as judged by 15N incorporation (Fig. 4d).

Bottom Line: However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation.Moreover, Gln-starved cells are not rescued by TCA cycle replenishment.In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons.

View Article: PubMed Central - PubMed

Affiliation: Cancer Metabolism Research Unit, Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

Show MeSH
Related in: MedlinePlus