Environmental cystine drives glutamine anaplerosis and sensitizes cancer cells to glutaminase inhibition
- Massachusetts Institute of Technology, United States
- Whitehead Institute for Biomedical Research, United States
- Dana-Farber Cancer Institute, United States
Figures
Figure 1 with 3 supplements
Decreased use of glutamine for TCA cycle anaplerosis by A549 cells in tumors, and when cultured in adult bovine serum, compared to RPMI.
(A) Diagram detailing how uniformly-labeled glutamine ([U-13C5]glutamine) can be metabolized to generate labeled glutamate, aspartate and TCA cycle intermediates via oxidative metabolism. (B) Left …
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Figure 1—source data 1
Mass isotopomer distributions for all metabolites analyzed by GC-MS in Figure 1.
- https://doi.org/10.7554/eLife.27713.007
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Figure 1—source data 2
Mass isotopomer distributions for all metabolites analyzed by GC-MS in Figure 1—figure supplement 1.
- https://doi.org/10.7554/eLife.27713.008
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Figure 1—source data 3
Mass isotopomer distributions for all metabolites analyzed by GC-MS in Figure 1—figure supplement 2.
- https://doi.org/10.7554/eLife.27713.009
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Figure 1—source data 4
Mass isotopomer distributions for all metabolites analyzed by GC-MS in Figure 1—figure supplement 3.
- https://doi.org/10.7554/eLife.27713.010
Figure 1—figure supplement 1
Glutamine labeling of metabolites in A549 cells cultured in adult bovine serum reaches isotopic steady state by 8 hr.
M + 5 fractional labeling of glutamine, glutamate and α-ketoglutarate, and m + 4 labeling of fumarate, malate, aspartate and citrate for A549 cells cultured for 8 hr or 24 hr in adult bovine serum …
Figure 1—figure supplement 2
Decreased glutamine anaplerosis is not unique to serum or bovine derived blood products.
M + 5 fractional labeling of glutamine, glutamate and α-ketoglutarate, and m + 4 labeling of fumarate, malate, aspartate and citrate for A549 cells cultured for 8 hr in RPMI, adult bovine serum, …
Figure 1—figure supplement 3
Decreased glutamine anaplerosis in adult bovine serum is not unique to A549 cells.
M + 5 fractional labeling of glutamine, glutamate and α-ketoglutarate, and m + 4 fractional labeling of fumarate, malate, aspartate and citrate for the indicated cell lines cultured for 8 hr in RPMI …
Figure 2 with 1 supplement
Differences in the small molecule (<3.5 kDa) fraction between RPMI and adult bovine serum account for differences in glutamine anaplerosis and sensitivity to glutaminase inhibition.
(A) Diagram detailing the generation of top ‘Adult bovine serum → RPMI’ and bottom ‘RPMI → adult bovine serum’. To make each dialyzed medium, 210 mL of RPMI or adult bovine serum was dialyzed twice …
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Figure 2—source data 1
Mass isotopomer distributions for all metabolites analyzed by GC-MS in Figure 2.
- https://doi.org/10.7554/eLife.27713.013
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Figure 2—source data 2
Mass isotopomer distributions for all metabolites analyzed by GC-MS in Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.27713.014
Figure 2—figure supplement 1
Differences in the small molecule (<3.5 kDa) fraction between DMEM and adult bovine serum account for differences in glutamine anaplerosis and sensitivity to glutaminase inhibition.
(A) Diagram detailing the generation of top ‘Adult bovine serum → DMEM’ and bottom ‘DMEM → adult bovine serum’. To make each dialyzed medium, 210 mL of DMEM or adult bovine serum was dialyzed twice …
Figure 3 with 2 supplements
High levels of cystine enhance glutamine anaplerosis and potentiate the effects of the glutaminase inhibitor CB-839.
(A) M + 5 fractional labeling of glutamine, glutamate and α-ketoglutarate, and m + 4 labeling of fumarate, malate, aspartate and citrate is shown for A549 cells cultured for 8 hr in RPMI, adult …
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Figure 3—source data 1
Mass isotopomer distributions for all metabolites analyzed by GC-MS in Figure 3.
- https://doi.org/10.7554/eLife.27713.019
Figure 3—figure supplement 1
Identification of cystine as the metabolite in standard culture media that potentiates the glutaminase inhibitor CB-839.
(A) A549 cells were cultured in adult bovine serum with DMEM nutrient levels, adult bovine serum with DMEM amino acid levels and adult bovine serum with DMEM amino acid levels, but without …
Figure 3—figure supplement 2
Glutathione depletion and ROS are not solely responsible for decreased cell proliferation following glutaminase inhibition in high cystine environments.
(A) Glutamate has multiple possible metabolic fates in cells, any of which may become limiting upon glutaminase inhibition. Two fates indicated by red arrows (TCA cycle anaplerosis and glutathione …
Figure 4 with 4 supplements
The cystine/glutamate antiporter xCT/SLC7A11 is necessary and sufficient for cystine induced glutamine anaplerosis and CB-839 sensitivity.
(A) System xc– is a plasma membrane antiporter composed of two polypeptides, xCT (encoded by SLC7A11) and 4F2hc/CD98 (encoded by SLC3A2), that exchanges intracellular glutamate for extracellular …
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Figure 4—source data 1
Mass isotopomer distributions for all metabolites analyzed by GC-MS in Figure 4.
Cell line identity and SLC7A11 mRNA expression level for cell lines analyzed in 4F. Cell line identity and CB-839 sensitivity for cell lines analyzed in 4G.
- https://doi.org/10.7554/eLife.27713.025
Figure 4—figure supplement 1
Extracellular cystine alters both cellular glutamine and cystine consumption and glutamate release rates.
(A) A549 cells were cultured in RPMI or RPMI with 10 µM cystine. Per cell rates of glutamine consumption, glutamate release and cystine consumption were measured as detailed in Materials and methods …
Figure 4—figure supplement 2
Knockdown of SLC7A11 reduces cellular glutamine uptake and glutamate release potentiated by high environmental cystine.
(A) A549 cells infected with lentiviruses encoding SLC7A11 targeting shRNAs or a control shRNA targeting GFP were cultured in RPMI or RPMI with 10 µM cystine. Per cell rates of glutamine consumption …
Figure 4—figure supplement 3
SLC7A11 expression enhances cellular glutamine uptake and glutamate release potentiated by high environmental cystine.
(A) MCF7 cells infected with lentiviruses encoding SLC7A11 cDNA or empty vector (E.V.). were cultured in RPMI or RPMI with 10 µM cystine. Per cell rates of glutamine consumption and glutamate …
Figure 4—figure supplement 4
Overexpression of xCT/SLC7A11 causes cystine-induced CB-839 sensitivity for MDA-MB-468 and AU565 breast cancer cell lines.
Proliferation rates for MDA-MB-468 and AU565 cell lines overexpressing SLC7A11 or empty vector (from Figure 4G) cultured in RPMI or RPMI with 10 µM cystine in the presence of vehicle (DMSO) or 1 µM …
Figure 5
Raising cystine levels increases glutamine metabolism in tumors.
(A) nu/nu mice were treated with 2.4 g/kg cystine by oral gavage, and plasma from these animals was collected at the indicated time points. Cystine concentration in plasma at each time point as …
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Figure 5—source data 1
Mass isotopomer distributions for all metabolites analyzed by GC-MS in Figure 5.
- https://doi.org/10.7554/eLife.27713.027
Tables
Table 1
Amino acid, glucose, pyruvate and lactate concentrations in all media used in this study compared to human plasma clinical reference values
https://doi.org/10.7554/eLife.27713.015| Metabolite | Human male plasma reference range* [µM] | RPMI-1640 with 10% dialyzed fetal bovine serum [µM] | DMEM with 10% dialyzed fetal bovine serum [µM] | Adult bovine serum† [µM] | Adult bovine heparinized plasma† [µM] | Adult human serum† [µM] |
|---|---|---|---|---|---|---|
| Alanine | 146–494 | 0 | 0 | 314 ± 6 | 321 ± 3 | 670 ± 13 |
| Arginine | 28–96 | 1034 | 360 | 312 ± 10 | 150 ± 9 | 216 ± 8 |
| Aspargine | 32–92 | 341 | 0 | 17 ± 1 | 19 ± 1 | 81 ± 2 |
| Aspartate | 2–9 | 135 | 0 | 7.4 ± 0.4 | 5.3 ± 0.4 | 59.2 ± 1.3 |
| Cystine | 24–54 | 187 | 180 | 0.3 ± 0.1 | 2 ± 0.1 | 3.4 ± 0.2 |
| Glutamate | 6–62 | 122 | 0 | 192 ± 3 | 120 ± 1 | 348 ± 5 |
| Glutamine | 466–798 | 1849 | 3600 | 183 ± 4 | 291 ± 1 | 409 ± 7 |
| Glycine | 147–299 | 120 | 360 | 302 ± 6 | 221 ± 1 | 409 ± 8 |
| Histidine | 72–108 | 87 | 180 | 8.9 ± 0.2 | 8.2 ± 0.2 | 17.7 ± 0.3 |
| Isoleucine | 46–90 | 344 | 720 | 112 ± 2 | 84 ± 1 | 114 ± 1 |
| Leucine | 113–205 | 344 | 720 | 218 ± 3 | 172 ± 1 | 231 ± 5 |
| Lysine | 135–243 | 197 | 720 | 92 ± 2 | 146 ± 1 | 206 ± 4 |
| Methionine | 13–37 | 91 | 180 | 21 ± 1 | 26 ± 1 | 39 ± 1 |
| Phenylalanine | 46–74 | 82 | 360 | 92 ± 2 | 68 ± 1 | 144 ± 3 |
| Proline | 97–297 | 157 | 0 | 88 ± 2 | 61 ± 1 | 301 ± 4 |
| Serine | 89–165 | 257 | 360 | 119 ± 2 | 74 ± 1 | 237 ± 5 |
| Threonine | 92–180 | 151 | 720 | 63 ± 1 | 50 ± 1 | 192 ± 3 |
| Tryptophan | 25–65 | 22 | 72 | ND | ND | ND |
| Tyrosine | 37–77 | 99 | 360 | 56 ± 1 | 47 ± 1 | 101 ± 3 |
| Valine | 179–335 | 154 | 720 | 251 ± 5 | 181 ± 2 | 325 ± 6 |
| Glucose | 9990 | 22500 | 3932 ± 26 | 8403 ± 33 | 2407 ± 23 | |
| Pyruvate | 27–160 | 0 | 900 | 9.4 ± 0.2 | 70.8 ± 0.5 | 10.3 ± 0.7 |
| Lactate | 0 | 0 | 10878 ± 217 | 9291 ± 61 | 9250 ± 178 |
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* Shown is the range ±2 standard deviations from the mean value for the indicated metabolite. These values are from (Blau, 2003).
† Shown are the mean values ± the standard error of the mean for the indicated metabolites as determined by GC-MS (see Materials and methods for detailed procedure), except glucose concentration, which was determined using a YSI bioanalyzer. All samples were analyzed in triplicate. ND indicates that the metabolite was not detected.
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.27713.028
