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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 GBM patients and primary orthotopic xenografts. (a) GBM tissue microarray. GS immuno-staining of representative tissue cores at low and high magnification (top and bottom respectively). A: Astrocyte, N: Neuron. (b) Frequency distribution of GBM patients (n=209) divided according to their histoscore for GS, and categorized as low, medium, and high. Normal astrocytes were used as a reference for defining maximal immunoreactivity. (c) Kaplan Meier curves for GBM patients divided into low, medium, and high GS expression. p value refers to a log-rank (Mantel-Cox) test. (d-e) creatine, choline, choline to creatine ratio (d), and Gln (e) levels in tumor tissue and adjacent edematous brain of GBM patients injected with 13C6-glucose before surgical intervention. n= 7 patients, p values refer to a two-tailed t test for paired samples. (f-g) 13C6-glucose (f), and 13C Gln (g) enrichment in serum at time of tumour resection, in tumor tissue, and in adjacent edematous tissue. Gln isotopologues incorporating one or more 13C-atoms, over the total amount of Gln detected (% of total) are shown. na: not available, nd: not detectable. Values were corrected for the natural abundance of 13C. (h) Coronal section of human P3 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Lower panels are magnification of the respective framed regions. A: astrocytes, AF: astrocytic end-feet, N: neuron, V: blood vessel. (i, j) Isotopologue distribution of metabolites (Hexose phosphates, citrate, α-KG, Glu, Gln) obtained in mice orthotopically xenografted with human P3 GBM, and injected in the tail vain with a bolus of 13C6-Glucose (i) or 13C5-Gln (j). Tissues were sampled 22min after injection. The values are mean ± S.E.M. n=3 mice for all conditions, except for contralateral brain of mice injected with glucose, where 2 mice were used. (i, j) Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Figure 6: Gln metabolism in GBM patients and primary orthotopic xenografts. (a) GBM tissue microarray. GS immuno-staining of representative tissue cores at low and high magnification (top and bottom respectively). A: Astrocyte, N: Neuron. (b) Frequency distribution of GBM patients (n=209) divided according to their histoscore for GS, and categorized as low, medium, and high. Normal astrocytes were used as a reference for defining maximal immunoreactivity. (c) Kaplan Meier curves for GBM patients divided into low, medium, and high GS expression. p value refers to a log-rank (Mantel-Cox) test. (d-e) creatine, choline, choline to creatine ratio (d), and Gln (e) levels in tumor tissue and adjacent edematous brain of GBM patients injected with 13C6-glucose before surgical intervention. n= 7 patients, p values refer to a two-tailed t test for paired samples. (f-g) 13C6-glucose (f), and 13C Gln (g) enrichment in serum at time of tumour resection, in tumor tissue, and in adjacent edematous tissue. Gln isotopologues incorporating one or more 13C-atoms, over the total amount of Gln detected (% of total) are shown. na: not available, nd: not detectable. Values were corrected for the natural abundance of 13C. (h) Coronal section of human P3 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Lower panels are magnification of the respective framed regions. A: astrocytes, AF: astrocytic end-feet, N: neuron, V: blood vessel. (i, j) Isotopologue distribution of metabolites (Hexose phosphates, citrate, α-KG, Glu, Gln) obtained in mice orthotopically xenografted with human P3 GBM, and injected in the tail vain with a bolus of 13C6-Glucose (i) or 13C5-Gln (j). Tissues were sampled 22min after injection. The values are mean ± S.E.M. n=3 mice for all conditions, except for contralateral brain of mice injected with glucose, where 2 mice were used. (i, j) Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Mentions: As shown above, GS activity, largely determining Gln dependency, varies between established and primary human GBM cells. Similarly, tissue microarray (TMA) analysis showed that GS expression varies between human GBM patients (n=209), resembling a Gaussian distribution ranging from tumours with low GS levels, comparable to neurons (25% of patients), to high-expression tumours comparable to astrocytes (15%) (Fig 6a-b). However, GS expression did not predict patient median survival (Fig. 6c). Of 20 biopsies, from which core TMA were sampled, 5 showed substantial intra-tumoral GS immunostaining heterogeneity (3 examples are reported in Supplementary Fig. 5). Most GBMs either showed GS uniformity, or a mosaic infiltration of GS-positive cells, suggesting autonomous intra-tumoral Gln biosynthetic capacity. To assess this hypothesis in human tumours, seven GBM patients were injected with 13C6-glucose prior to surgery, and metabolites were extracted from the resected tumours and their edematous margins. The metabolic analysis reliably discriminated between tumour and adjacent tissues by using the choline to creatine ratio, a parameter for classifying brain tumours by MR spectroscopy32 (Fig. 6d). No significant difference in Gln content was observed between tumour and adjacent tissues (Fig. 6e). At the time of resection, 13C6-glucose enrichment in serums ranged between 16% and 50% (Fig. 6f, and Supplementary Fig. 6a). Glucose-derived 13C-Gln was detected in 6/7 tumours and in 7/7 adjacent edematous tissues with an enrichment ranging between 1% and 12% (Fig. 6g). In 3/5 patients the fraction of glucose-derived Gln in the tumour was higher than in the serum sample, and so not in equilibrium with the circulating Gln, suggesting that the tumour Gln pool is synthesized in situ and/or provided by adjacent normal brain.


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 GBM patients and primary orthotopic xenografts. (a) GBM tissue microarray. GS immuno-staining of representative tissue cores at low and high magnification (top and bottom respectively). A: Astrocyte, N: Neuron. (b) Frequency distribution of GBM patients (n=209) divided according to their histoscore for GS, and categorized as low, medium, and high. Normal astrocytes were used as a reference for defining maximal immunoreactivity. (c) Kaplan Meier curves for GBM patients divided into low, medium, and high GS expression. p value refers to a log-rank (Mantel-Cox) test. (d-e) creatine, choline, choline to creatine ratio (d), and Gln (e) levels in tumor tissue and adjacent edematous brain of GBM patients injected with 13C6-glucose before surgical intervention. n= 7 patients, p values refer to a two-tailed t test for paired samples. (f-g) 13C6-glucose (f), and 13C Gln (g) enrichment in serum at time of tumour resection, in tumor tissue, and in adjacent edematous tissue. Gln isotopologues incorporating one or more 13C-atoms, over the total amount of Gln detected (% of total) are shown. na: not available, nd: not detectable. Values were corrected for the natural abundance of 13C. (h) Coronal section of human P3 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Lower panels are magnification of the respective framed regions. A: astrocytes, AF: astrocytic end-feet, N: neuron, V: blood vessel. (i, j) Isotopologue distribution of metabolites (Hexose phosphates, citrate, α-KG, Glu, Gln) obtained in mice orthotopically xenografted with human P3 GBM, and injected in the tail vain with a bolus of 13C6-Glucose (i) or 13C5-Gln (j). Tissues were sampled 22min after injection. The values are mean ± S.E.M. n=3 mice for all conditions, except for contralateral brain of mice injected with glucose, where 2 mice were used. (i, j) Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Figure 6: Gln metabolism in GBM patients and primary orthotopic xenografts. (a) GBM tissue microarray. GS immuno-staining of representative tissue cores at low and high magnification (top and bottom respectively). A: Astrocyte, N: Neuron. (b) Frequency distribution of GBM patients (n=209) divided according to their histoscore for GS, and categorized as low, medium, and high. Normal astrocytes were used as a reference for defining maximal immunoreactivity. (c) Kaplan Meier curves for GBM patients divided into low, medium, and high GS expression. p value refers to a log-rank (Mantel-Cox) test. (d-e) creatine, choline, choline to creatine ratio (d), and Gln (e) levels in tumor tissue and adjacent edematous brain of GBM patients injected with 13C6-glucose before surgical intervention. n= 7 patients, p values refer to a two-tailed t test for paired samples. (f-g) 13C6-glucose (f), and 13C Gln (g) enrichment in serum at time of tumour resection, in tumor tissue, and in adjacent edematous tissue. Gln isotopologues incorporating one or more 13C-atoms, over the total amount of Gln detected (% of total) are shown. na: not available, nd: not detectable. Values were corrected for the natural abundance of 13C. (h) Coronal section of human P3 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Lower panels are magnification of the respective framed regions. A: astrocytes, AF: astrocytic end-feet, N: neuron, V: blood vessel. (i, j) Isotopologue distribution of metabolites (Hexose phosphates, citrate, α-KG, Glu, Gln) obtained in mice orthotopically xenografted with human P3 GBM, and injected in the tail vain with a bolus of 13C6-Glucose (i) or 13C5-Gln (j). Tissues were sampled 22min after injection. The values are mean ± S.E.M. n=3 mice for all conditions, except for contralateral brain of mice injected with glucose, where 2 mice were used. (i, j) Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
Mentions: As shown above, GS activity, largely determining Gln dependency, varies between established and primary human GBM cells. Similarly, tissue microarray (TMA) analysis showed that GS expression varies between human GBM patients (n=209), resembling a Gaussian distribution ranging from tumours with low GS levels, comparable to neurons (25% of patients), to high-expression tumours comparable to astrocytes (15%) (Fig 6a-b). However, GS expression did not predict patient median survival (Fig. 6c). Of 20 biopsies, from which core TMA were sampled, 5 showed substantial intra-tumoral GS immunostaining heterogeneity (3 examples are reported in Supplementary Fig. 5). Most GBMs either showed GS uniformity, or a mosaic infiltration of GS-positive cells, suggesting autonomous intra-tumoral Gln biosynthetic capacity. To assess this hypothesis in human tumours, seven GBM patients were injected with 13C6-glucose prior to surgery, and metabolites were extracted from the resected tumours and their edematous margins. The metabolic analysis reliably discriminated between tumour and adjacent tissues by using the choline to creatine ratio, a parameter for classifying brain tumours by MR spectroscopy32 (Fig. 6d). No significant difference in Gln content was observed between tumour and adjacent tissues (Fig. 6e). At the time of resection, 13C6-glucose enrichment in serums ranged between 16% and 50% (Fig. 6f, and Supplementary Fig. 6a). Glucose-derived 13C-Gln was detected in 6/7 tumours and in 7/7 adjacent edematous tissues with an enrichment ranging between 1% and 12% (Fig. 6g). In 3/5 patients the fraction of glucose-derived Gln in the tumour was higher than in the serum sample, and so not in equilibrium with the circulating Gln, suggesting that the tumour Gln pool is synthesized in situ and/or provided by adjacent normal brain.

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