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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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Glutamine supply for GBM tumours with low GS expression. (a) Gln and Asn levels were measured by HPLC-MS in peripheral blood samples obtained at indicated time points from immunocompromised mice intraperitoneally injected with Erwinase (5U/gr of body weight). Mean ± S.E.M. n=5 mice. p values refer to a two-tailed t test for paired samples. (b) Coronal section of human T101 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Left panels are magnification of the respective framed regions. A: astrocytes, N: neuron. (c) Immunocompromised mice were orthotopically implanted with T101 GBM tumours and treated with Erwinase for 6 weeks as described in the Methods section. Asn and Gln were assessed in the tumour and contralateral brain 6h after the last Erwinase injection. Mean ± S.E.M. n=3 mice. (d) MRI-based Apparent Diffusion Coefficient (ADC) maps of T101 GBM tumours treated with Erwinase as described in (c). The tumour mask has been manually delineated to highlight the tumour region. IHC staining of brain sections corresponding to the MRI scans are shown. T101 tumours were stained with an anti-human EGFR antibody. (e) Two representative series of coronal sections of T101 brain xenografts, were stained for human EGFR. Mice were treated with Erwinase as in (c). (f) Volumes of T101 orthotopic tumours obtained thorough quantitative imaging of EGFR-stained serial sections of brains. Mice were treated with Erwinase as in (c). Mean ± S.E.M. n=7 mice. p value refer to a two-tailed t test for unpaired samples. (g) Coronal section of human T407 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Left panels are magnification of the respective framed regions. A: astrocytes, V: blood vessel. (h) 15N1-Gln enrichment in T407 GBM tumours and in contralateral brains, after a 4h intracarotid infusion with . The dashed lines correspond to the natural abundance of 15N1-Gln. Mean ± S.E.M. n=4 mice. p value refer to a two-tailed t test for paired samples.
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Figure 7: Glutamine supply for GBM tumours with low GS expression. (a) Gln and Asn levels were measured by HPLC-MS in peripheral blood samples obtained at indicated time points from immunocompromised mice intraperitoneally injected with Erwinase (5U/gr of body weight). Mean ± S.E.M. n=5 mice. p values refer to a two-tailed t test for paired samples. (b) Coronal section of human T101 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Left panels are magnification of the respective framed regions. A: astrocytes, N: neuron. (c) Immunocompromised mice were orthotopically implanted with T101 GBM tumours and treated with Erwinase for 6 weeks as described in the Methods section. Asn and Gln were assessed in the tumour and contralateral brain 6h after the last Erwinase injection. Mean ± S.E.M. n=3 mice. (d) MRI-based Apparent Diffusion Coefficient (ADC) maps of T101 GBM tumours treated with Erwinase as described in (c). The tumour mask has been manually delineated to highlight the tumour region. IHC staining of brain sections corresponding to the MRI scans are shown. T101 tumours were stained with an anti-human EGFR antibody. (e) Two representative series of coronal sections of T101 brain xenografts, were stained for human EGFR. Mice were treated with Erwinase as in (c). (f) Volumes of T101 orthotopic tumours obtained thorough quantitative imaging of EGFR-stained serial sections of brains. Mice were treated with Erwinase as in (c). Mean ± S.E.M. n=7 mice. p value refer to a two-tailed t test for unpaired samples. (g) Coronal section of human T407 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Left panels are magnification of the respective framed regions. A: astrocytes, V: blood vessel. (h) 15N1-Gln enrichment in T407 GBM tumours and in contralateral brains, after a 4h intracarotid infusion with . The dashed lines correspond to the natural abundance of 15N1-Gln. Mean ± S.E.M. n=4 mice. p value refer to a two-tailed t test for paired samples.

Mentions: Next, Erwinase, an enzyme that temporarily depletes circulating asparagine (Asn) and significantly reduces Gln9,33 (Fig 7a), was injected daily (five times per week) into mice bearing GS-negative orthotopic GBM xenografts (T101, Fig. 7b). Erwinase reduced intra-tumoral and intracerebral Asn but not Gln (Fig. 7c). The diffuse morphology of GS-negative tumours impaired Ex vivo MRI analysis volumetric quantification (Fig. 7d). Histological reconstruction to assess tumour burden (Fig. 7e) revealed no significant differences between control and Erwinase treated groups (Fig. 7f). Altogether, these results indicate that Gln is not provided to GBM by the blood and so, the proximity of GS-positive astrocytes and GS-negative glioma cells (Fig. 7b and 7g as representatives) suggest that astrocytes may be a source of Gln. Indeed, when mice bearing GS-negative GBM xenografts (T407, Fig 7g) were infused with 15N1-ammonia into the carotid artery for 4 hours, the fraction of 15N-Gln was ~5% in both tumour and contralateral brain tissues (Fig 7h) indicating that they are a secluded, autonomous compartment for Gln biosynthesis and utilization, where GS expressing cells supply Gln to GS-negative ones.


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)

Glutamine supply for GBM tumours with low GS expression. (a) Gln and Asn levels were measured by HPLC-MS in peripheral blood samples obtained at indicated time points from immunocompromised mice intraperitoneally injected with Erwinase (5U/gr of body weight). Mean ± S.E.M. n=5 mice. p values refer to a two-tailed t test for paired samples. (b) Coronal section of human T101 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Left panels are magnification of the respective framed regions. A: astrocytes, N: neuron. (c) Immunocompromised mice were orthotopically implanted with T101 GBM tumours and treated with Erwinase for 6 weeks as described in the Methods section. Asn and Gln were assessed in the tumour and contralateral brain 6h after the last Erwinase injection. Mean ± S.E.M. n=3 mice. (d) MRI-based Apparent Diffusion Coefficient (ADC) maps of T101 GBM tumours treated with Erwinase as described in (c). The tumour mask has been manually delineated to highlight the tumour region. IHC staining of brain sections corresponding to the MRI scans are shown. T101 tumours were stained with an anti-human EGFR antibody. (e) Two representative series of coronal sections of T101 brain xenografts, were stained for human EGFR. Mice were treated with Erwinase as in (c). (f) Volumes of T101 orthotopic tumours obtained thorough quantitative imaging of EGFR-stained serial sections of brains. Mice were treated with Erwinase as in (c). Mean ± S.E.M. n=7 mice. p value refer to a two-tailed t test for unpaired samples. (g) Coronal section of human T407 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Left panels are magnification of the respective framed regions. A: astrocytes, V: blood vessel. (h) 15N1-Gln enrichment in T407 GBM tumours and in contralateral brains, after a 4h intracarotid infusion with . The dashed lines correspond to the natural abundance of 15N1-Gln. Mean ± S.E.M. n=4 mice. p value refer to a two-tailed t test for paired samples.
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Figure 7: Glutamine supply for GBM tumours with low GS expression. (a) Gln and Asn levels were measured by HPLC-MS in peripheral blood samples obtained at indicated time points from immunocompromised mice intraperitoneally injected with Erwinase (5U/gr of body weight). Mean ± S.E.M. n=5 mice. p values refer to a two-tailed t test for paired samples. (b) Coronal section of human T101 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Left panels are magnification of the respective framed regions. A: astrocytes, N: neuron. (c) Immunocompromised mice were orthotopically implanted with T101 GBM tumours and treated with Erwinase for 6 weeks as described in the Methods section. Asn and Gln were assessed in the tumour and contralateral brain 6h after the last Erwinase injection. Mean ± S.E.M. n=3 mice. (d) MRI-based Apparent Diffusion Coefficient (ADC) maps of T101 GBM tumours treated with Erwinase as described in (c). The tumour mask has been manually delineated to highlight the tumour region. IHC staining of brain sections corresponding to the MRI scans are shown. T101 tumours were stained with an anti-human EGFR antibody. (e) Two representative series of coronal sections of T101 brain xenografts, were stained for human EGFR. Mice were treated with Erwinase as in (c). (f) Volumes of T101 orthotopic tumours obtained thorough quantitative imaging of EGFR-stained serial sections of brains. Mice were treated with Erwinase as in (c). Mean ± S.E.M. n=7 mice. p value refer to a two-tailed t test for unpaired samples. (g) Coronal section of human T407 GBM xenograft grown in brain of immunocompromised mice, and stained for human nestin and GS. Left panels are magnification of the respective framed regions. A: astrocytes, V: blood vessel. (h) 15N1-Gln enrichment in T407 GBM tumours and in contralateral brains, after a 4h intracarotid infusion with . The dashed lines correspond to the natural abundance of 15N1-Gln. Mean ± S.E.M. n=4 mice. p value refer to a two-tailed t test for paired samples.
Mentions: Next, Erwinase, an enzyme that temporarily depletes circulating asparagine (Asn) and significantly reduces Gln9,33 (Fig 7a), was injected daily (five times per week) into mice bearing GS-negative orthotopic GBM xenografts (T101, Fig. 7b). Erwinase reduced intra-tumoral and intracerebral Asn but not Gln (Fig. 7c). The diffuse morphology of GS-negative tumours impaired Ex vivo MRI analysis volumetric quantification (Fig. 7d). Histological reconstruction to assess tumour burden (Fig. 7e) revealed no significant differences between control and Erwinase treated groups (Fig. 7f). Altogether, these results indicate that Gln is not provided to GBM by the blood and so, the proximity of GS-positive astrocytes and GS-negative glioma cells (Fig. 7b and 7g as representatives) suggest that astrocytes may be a source of Gln. Indeed, when mice bearing GS-negative GBM xenografts (T407, Fig 7g) were infused with 15N1-ammonia into the carotid artery for 4 hours, the fraction of 15N-Gln was ~5% in both tumour and contralateral brain tissues (Fig 7h) indicating that they are a secluded, autonomous compartment for Gln biosynthesis and utilization, where GS expressing cells supply Gln to GS-negative ones.

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