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. 2017 Jul 6;67(1):128-138.e7.
doi: 10.1016/j.molcel.2017.05.030. Epub 2017 Jun 22.

mTORC2 Regulates Amino Acid Metabolism in Cancer by Phosphorylation of the Cystine-Glutamate Antiporter xCT

Yuchao Gu  1 , Claudio P Albuquerque  2 , Daniel Braas  3 , Wei Zhang  4 , Genaro R Villa  5 , Junfeng Bi  2 , Shiro Ikegami  6 , Kenta Masui  7 , Beatrice Gini  8 , Huijun Yang  2 , Timothy C Gahman  9 , Andrew K Shiau  9 , Timothy F Cloughesy  10 , Heather R Christofk  3 , Huilin Zhou  11 , Kun-Liang Guan  12 , Paul S Mischel  13
Affiliations

mTORC2 Regulates Amino Acid Metabolism in Cancer by Phosphorylation of the Cystine-Glutamate Antiporter xCT

Yuchao Gu et al. Mol Cell. .

Abstract

Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic inhibition of the mammalian target of rapamycin (mTOR) kinase, promotes glutamate secretion, cystine uptake, and incorporation into glutathione, linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism regulating amino acid metabolism in cancer, enabling tumor cells to adapt to changing environmental conditions.

Keywords: cancer; glioblastoma; glutamate metabolism; glutathione metabolism; mTORC2; xCT.

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Figures

Figure 1
Figure 1. xCT physically interacts with mTOR Complex 2 in GBM cells
(A) A brief schematic of the SILAC labeling and Mass Spectrometry experiment performed to identify xCT specific binding proteins in U87EGFRvIII cells. (B) The median fold enrichment of the identified proteins was plotted on a Log10 scale as xCT versus vector. A cutoff of Log10 (xCT/vector) <1 was applied and indicated by the shaded area below the dash line. Known xCT binding proteins as well as mTOR and Rictor were labeled in red. (C) DAVID gene ontology (GO) analysis was performed using the list of 125 potential xCT binding proteins identified in (B). Top 10 enriched biological pathways were plotted using the – (Log10 FDR). The enriched pathway that contain both mTOR and Rictor were indicated in red and the full gene list of each pathway can be found in Table S2. (D) Co-immunoprecipitation (Co-IP) was performed to validate mTOR and Rictor as xCT binding proteins in GBM (T98G), breast cancer (MDA-MB-231, Hs578T), and lung cancer (A549) cell lines stably expressing the FLAG-tagged xCT or vector control. See also Figure S1, Table S1 and Table S2.
Figure 2
Figure 2. mTORC2 phosphorylates xCT downstream of growth factor signaling
(A) RXXS/T motifs on xCT were listed by analyzing xCT protein sequence. S26 (in red) phosphorylation was detected in our study and has been reported by others. S51 and S481 (in blue) phosphorylation were reported on PhosphoSitePlus but were not detected in our experiments. Phosphorylation of the remaining RXXS/T sites (in black) on xCT has not been reported in any other studies or observed in our experiments (Hornbeck et al., 2015). (http://www.phosphosite.org/uniprotAccAction?id=Q9UPY5.) (B) Immunoprecipitation (IP) - western blot was performed in U87 cells stably expressing wtEGFR and myc-tagged xCT. Cells were serum starved for 24 h post 24 h of transfection with siRNA before stimulation with 25 ng/ml EGF. Cell lysates were collected at indicated time points and subjected to pRXXS/T IP and western blotting analysis. (C) U87EGFRvIII cells stably overexpressing xCT or vector control were treated with 250 nM Torin1. Protein lysates were collected over a time course of 24 h for IP-western blot to determine xCT phosphorylation on RXXS/T motifs. See also Figure S2.
Figure 3
Figure 3. mTORC2 phosphorylates xCT on serine26
(A) A simplified schematic diagram of xCT 2D structure constructed based on sequence and predicted domains of xCT obtained from UniProt-KB. Transmembrane domains were shown as cylinders. Potential phosphorylation sites within RXXS/T motifs were labeled with the same color code as in Fig.2A. (B) A simplified schematic diagram depicting xCT mutants generated and used in the following experiments. (C) Phosphorylation on RXXS/T motifs in wildtype and mutant xCT were analyzed by pRXXS/T IP and western blot. (D) Phosphorylation on xCT serine 26 is conserved across species. (Hornbeck et al., 2015). (http://www.phosphosite.org/uniprotAccAction?id=Q9UPY5.) (E) Schematic of LC-MS/MS to identify potential phosphorylation sites on xCT in GBM cells. (F) Tandem mass spectrometry (MS/MS) spectra showing phosphorylation of xCT on serine 26 in U87EGFRvIII cells. (G) In vitro kinase assay was carried out by incubating mTORC2 IP-purified from HEK293T cells, peptide substrates and [γ-32P]-ATP in kinase reaction buffer at room temperature for 1 h. Scintillation counts from three independent replicates were presented as mean counts per minute (cpm) ± SEM. Statistical analysis was performed using one-way ANOVA. *** refers to p value < 0.001. n.s. refers to not statistically significant. (H) IP-western blot detecting wild-type or S26A mutant xCT phosphorylation on RXXS/T motifs upon EGF stimulation. See also Figure S3.
Figure 4
Figure 4. Inhibition of xCT phosphorylation on serine 26 increases glutamate/cystine transport and supports glutathione synthesis
(A) Cell surface and total protein levels of xCT were analyzed by western blotting in xCT KO MEFs stably overexpressing vector control, wt xCT and xCT S26A. Band intensities were quantified by densitometry and relative surface xCT levels was calculated, and normalized to Na, K-ATPase as loading control of cell surface proteins. (B) xCT activity assay was performed in xCT KO MEFs stably overexpressing vector control, wt xCT and xCT S26A. Glutamate secretion was measured using the AmplexRed glutamate assay kit, calculated and normalized to cell counts and cell surface xCT protein levels. Results were obtained from three replicates and data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *** refers to p value < 0.001. (C) Glutamate secretion was measured using the AmplexRed glutamate assay kit in U87EGFRvIII cells with stable Rictor knockdown using two different shRNAs. Results were obtained from three independent replicates and data are presented as mean ± SEM. *** refers to p value < 0.001. Statistical analysis was performed using two-way ANOVA comparing the mean of shRictor1 and shRictor2 to shscramble control. (D) Glutamate secretion was measured in U87EGFRvIII cells treated with Torin1 for 24 h after transfected with 50 nM siRNA targeting xCT for 48 h by NOVA Bioprofile 400 analyzer. Results were obtained from three independent replicates and data are presented as mean ± SEM. **refers to p value < 0.01. n.s. refers to not statistically significant. Statistical analysis was performed using two-way ANOVA comparing the mean of Torin1 to DMSO control in each conditions. (E) Cystine uptake was measured in U87EGFRvIII cells after treatment with 250 nM Torin1 for 24 h using a sodium cyanide and sodium nitroprusside based assay (Egea et al., 2015; Nakagawa and Coe, 1999). Results were obtained from seven replicates and data are presented as mean ± SEM. *** refers to p value < 0.001. (F) A brief schematic showing the labeling process of GSH and GSSG with [3,3’-13C2] L-Cystine. (G) Exogenous cystine incorporation into newly synthesized glutathione was determined by labeling cells with [3,3’-13C2] L-Cystine together with 24 h of DMSO or Torin1 treatment in DMEM supplemented with 5% dialyzed FBS. The labeling percentage of both GSH and GSSG by [3,3’-13C2] L-Cystine were calculated. Each column represents three replicate samples collected at each time point. Data are presented as mean ± SEM. *** refers to p value < 0.001. n.s. refers to not statistically significant. (H) Total cellular GSH levels were measured using the GSH/GSSG-Glo™ Glutathione Assay Kit (Promega). U87EGFRvIII cells were treated with DMSO or 250 nM Torin1 for 24 h after being transfected with xCT siRNA for 48 h. Total cellular GSH levels were normalized to blank control and cell counts. Results were obtained from three replicate samples and data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *** refers to p value < 0.001. (I) U87EGFRvIII cells were treated with DMSO or 250 nM Torin1 with or without µM erastin which has been reported to induce ferroptosis in several cancer cell lines. Cells were collected and stained with FITC-Annexin V and PI after 24 h of treatment and cell death was analyzed by flow cytometry. %Ferroptotic cells shown in the bar graph on the right was calculated by adding up the percentage of cells in the upper and lower right quadrant in each graph on the left. See also Figure S4.
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