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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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GS sustains cell growth during Gln starvation. (a) GS and GLS catalysed reactions. (b) Cells were incubated for 24h +/− Gln and protein expression was assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. (c) Scatter plot of GS protein expression observed in Gln-fed condition (Arbitrary Units, AU) in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (d) LN18 and SF188 cells were incubated for 24h +/− Gln in the presence of 0.8 mM . The intracellular levels of Gln isotopologues are shown as % of 15N0-Gln in LN18 cells. Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5. (e) SF188 and U251 cells were incubated for 72h +/− Gln in medium supplemented with 4mM Glu and 1mM MSO as indicated. Cells numbers are shown as % of untreated control. Mean ± S.E.M. n=3 independent experiments. (f) SF188 and U251 cells stably expressing a non-targeting control shRNA (shNTC) or two sequences targeting GS (shGS-1 and shGS-2) were incubated for 24h +/− Gln. (g) Cells were incubated for 72h +/− Gln in medium supplemented with 4mM Glu, and 0.8mM , as indicated. Cell number is shown as % of the respective Gln-fed control. Mean ± S.E.M. n=3 independent experiments. (h) Cells were incubated for 12-17 days +/− Gln in medium supplemented with 4mM Glu, and 0.8mM  as indicated. Colonies obtained in representative wells are shown. n=4 independent experiments, quantified as shown in Supplementary Figure 4b.
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Figure 3: GS sustains cell growth during Gln starvation. (a) GS and GLS catalysed reactions. (b) Cells were incubated for 24h +/− Gln and protein expression was assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. (c) Scatter plot of GS protein expression observed in Gln-fed condition (Arbitrary Units, AU) in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (d) LN18 and SF188 cells were incubated for 24h +/− Gln in the presence of 0.8 mM . The intracellular levels of Gln isotopologues are shown as % of 15N0-Gln in LN18 cells. Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5. (e) SF188 and U251 cells were incubated for 72h +/− Gln in medium supplemented with 4mM Glu and 1mM MSO as indicated. Cells numbers are shown as % of untreated control. Mean ± S.E.M. n=3 independent experiments. (f) SF188 and U251 cells stably expressing a non-targeting control shRNA (shNTC) or two sequences targeting GS (shGS-1 and shGS-2) were incubated for 24h +/− Gln. (g) Cells were incubated for 72h +/− Gln in medium supplemented with 4mM Glu, and 0.8mM , as indicated. Cell number is shown as % of the respective Gln-fed control. Mean ± S.E.M. n=3 independent experiments. (h) Cells were incubated for 12-17 days +/− Gln in medium supplemented with 4mM Glu, and 0.8mM as indicated. Colonies obtained in representative wells are shown. n=4 independent experiments, quantified as shown in Supplementary Figure 4b.

Mentions: Together, GS and GLS control Gln homeostasis by catalysing opposite reactions (Fig. 2j and 3a). The mRNA levels of GS and GLS in all cell lines revealed no pattern of Gln dependency (Supplementary Fig. 4a). While it has been proposed that c-Myc determines Gln addiction by increasing GLS expression3,23,24, MYC-induced lung tumours were shown to increase GS expression2. In line with this, SF188 cells, harboring MYC amplification25, and expressing high levels of c-Myc (Fig. 3b and supplementary Fig. 4a), showed the highest levels of GS mRNA and protein (Fig. 3b and Supplementary Fig. 4a). In most cell lines, GS protein levels rose upon Gln deprivation (Fig. 3b). Furthermore, GS protein levels tend to increase in cells with decreased sensitivity to Gln withdrawal (Fig. 3c). Nevertheless, GS did not match the residual low amount of Gln found in Gln-starved cells (Fig. 2c). To investigate this apparent discrepancy, the metabolic flux via GS was assessed by the incorporation of 15N-labeled ammonia () into Gln in LN18 and SF188 cells, which display low and high levels of GS, respectively. Significant levels of 15N-labeled Gln were indeed detected in SF188 but not in LN18 cells (Fig. 3d). Next, SF188 and U251 cells, displaying high levels of GS and low sensitivity to Gln withdrawal, were incubated with L-methionine sulfoximine (MSO), a selective irreversible inhibitor of GS. MSO sensitized cells to Gln starvation and, further, abolished the protective effect of Glu supplementation (Fig. 3e). To complement this approach, GS expression was stably silenced in these cells by two shRNA sequences (Fig. 3f). Upon Gln starvation cell proliferation (Fig. 3g) as well as colony formation (Fig. 3h and Supplementary Fig. 4b) was lowered by GS silencing. Supplementation with the GS substrates Glu and ammonia, rescued Gln-deprived control cells more effectively than GS-silenced cells.


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)

GS sustains cell growth during Gln starvation. (a) GS and GLS catalysed reactions. (b) Cells were incubated for 24h +/− Gln and protein expression was assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. (c) Scatter plot of GS protein expression observed in Gln-fed condition (Arbitrary Units, AU) in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (d) LN18 and SF188 cells were incubated for 24h +/− Gln in the presence of 0.8 mM . The intracellular levels of Gln isotopologues are shown as % of 15N0-Gln in LN18 cells. Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5. (e) SF188 and U251 cells were incubated for 72h +/− Gln in medium supplemented with 4mM Glu and 1mM MSO as indicated. Cells numbers are shown as % of untreated control. Mean ± S.E.M. n=3 independent experiments. (f) SF188 and U251 cells stably expressing a non-targeting control shRNA (shNTC) or two sequences targeting GS (shGS-1 and shGS-2) were incubated for 24h +/− Gln. (g) Cells were incubated for 72h +/− Gln in medium supplemented with 4mM Glu, and 0.8mM , as indicated. Cell number is shown as % of the respective Gln-fed control. Mean ± S.E.M. n=3 independent experiments. (h) Cells were incubated for 12-17 days +/− Gln in medium supplemented with 4mM Glu, and 0.8mM  as indicated. Colonies obtained in representative wells are shown. n=4 independent experiments, quantified as shown in Supplementary Figure 4b.
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Figure 3: GS sustains cell growth during Gln starvation. (a) GS and GLS catalysed reactions. (b) Cells were incubated for 24h +/− Gln and protein expression was assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. (c) Scatter plot of GS protein expression observed in Gln-fed condition (Arbitrary Units, AU) in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (d) LN18 and SF188 cells were incubated for 24h +/− Gln in the presence of 0.8 mM . The intracellular levels of Gln isotopologues are shown as % of 15N0-Gln in LN18 cells. Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5. (e) SF188 and U251 cells were incubated for 72h +/− Gln in medium supplemented with 4mM Glu and 1mM MSO as indicated. Cells numbers are shown as % of untreated control. Mean ± S.E.M. n=3 independent experiments. (f) SF188 and U251 cells stably expressing a non-targeting control shRNA (shNTC) or two sequences targeting GS (shGS-1 and shGS-2) were incubated for 24h +/− Gln. (g) Cells were incubated for 72h +/− Gln in medium supplemented with 4mM Glu, and 0.8mM , as indicated. Cell number is shown as % of the respective Gln-fed control. Mean ± S.E.M. n=3 independent experiments. (h) Cells were incubated for 12-17 days +/− Gln in medium supplemented with 4mM Glu, and 0.8mM as indicated. Colonies obtained in representative wells are shown. n=4 independent experiments, quantified as shown in Supplementary Figure 4b.
Mentions: Together, GS and GLS control Gln homeostasis by catalysing opposite reactions (Fig. 2j and 3a). The mRNA levels of GS and GLS in all cell lines revealed no pattern of Gln dependency (Supplementary Fig. 4a). While it has been proposed that c-Myc determines Gln addiction by increasing GLS expression3,23,24, MYC-induced lung tumours were shown to increase GS expression2. In line with this, SF188 cells, harboring MYC amplification25, and expressing high levels of c-Myc (Fig. 3b and supplementary Fig. 4a), showed the highest levels of GS mRNA and protein (Fig. 3b and Supplementary Fig. 4a). In most cell lines, GS protein levels rose upon Gln deprivation (Fig. 3b). Furthermore, GS protein levels tend to increase in cells with decreased sensitivity to Gln withdrawal (Fig. 3c). Nevertheless, GS did not match the residual low amount of Gln found in Gln-starved cells (Fig. 2c). To investigate this apparent discrepancy, the metabolic flux via GS was assessed by the incorporation of 15N-labeled ammonia () into Gln in LN18 and SF188 cells, which display low and high levels of GS, respectively. Significant levels of 15N-labeled Gln were indeed detected in SF188 but not in LN18 cells (Fig. 3d). Next, SF188 and U251 cells, displaying high levels of GS and low sensitivity to Gln withdrawal, were incubated with L-methionine sulfoximine (MSO), a selective irreversible inhibitor of GS. MSO sensitized cells to Gln starvation and, further, abolished the protective effect of Glu supplementation (Fig. 3e). To complement this approach, GS expression was stably silenced in these cells by two shRNA sequences (Fig. 3f). Upon Gln starvation cell proliferation (Fig. 3g) as well as colony formation (Fig. 3h and Supplementary Fig. 4b) was lowered by GS silencing. Supplementation with the GS substrates Glu and ammonia, rescued Gln-deprived control cells more effectively than GS-silenced cells.

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