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. 2013 Oct 14;24(4):450-65.
doi: 10.1016/j.ccr.2013.08.020. Epub 2013 Oct 3.

Glutamine Sensitivity Analysis Identifies the xCT Antiporter as a Common Triple-Negative Breast Tumor Therapeutic Target

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Glutamine Sensitivity Analysis Identifies the xCT Antiporter as a Common Triple-Negative Breast Tumor Therapeutic Target

Luika A Timmerman et al. Cancer Cell. .
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Abstract

A handful of tumor-derived cell lines form the mainstay of cancer therapeutic development, yielding drugs with an impact typically measured as months to disease progression. To develop more effective breast cancer therapeutics and more readily understand their clinical impact, we constructed a functional metabolic portrait of 46 independently derived breast cell lines. Our analysis of glutamine uptake and dependence identified a subset of triple-negative samples that are glutamine auxotrophs. Ambient glutamine indirectly supports environmental cystine acquisition via the xCT antiporter, which is expressed on one-third of triple-negative tumors in vivo. xCT inhibition with the clinically approved anti-inflammatory sulfasalazine decreases tumor growth, revealing a therapeutic target in breast tumors of poorest prognosis and a lead compound for rapid, effective drug development.

Conflict of interest statement

We declare no conflicts of interest.

Figures

Figure 1
Figure 1. Nutrient Consumption in Breast-Derived Cell Lines
Icon codes in figure keys; dotted lines bracket proliferating, non-tumorigenic sample values. Icons represent mean values +/− SD. (A) Comparison of two nomenclatures used to describe samples in our study. Sample numbers of each type indicated. (B) Fluorescence increase of cell lines after 4 hour culture with 2-NBDG or 6-NBDG -labeled glucose relative to unlabeled cultures. (C) Population doubling times, calculated from standard growth curves. (D) Glucose uptake at 4 hrs (from part B) vs. glutamine consumption, derived from media depletion at 24 hours culture, normalized to cell number. (E) Amino acid consumption by 4 claudin low samples that consume little glucose or glutamine compared to HMECd samples. Values derived as in D; standard three letter amino acid codes in key. (F) Glutamine consumption at 24 hrs vs. hGAC GeneChip hybridization signal. Green squares on y-axis are the average hGAC signals from 3 CD10+, and 3 BerEP4+ purified normal breast samples. (G) Differential hGAC expression by basal vs. luminal or ER+ vs. ER samples in 8 clinical breast tumor datasets, downloaded from NCBI GEO and Chin 2006 (Chin et al., 2006). t-test p-values below paired box plots. See also Figure S1, and Tables S1, S2, S3.
Figure 2
Figure 2. Glutamine Restriction Slows Culture Expansion
Icon codes in figure keys; dotted lines bracket non-tumorigenic sample values; C, complete media; Q-, glutamine-free media; U-, glucose free media. Icons represent mean values +/−SD. (A) Day 5 culture sizes for each cell line grown in glutamine-free media, normalized to culture in control media. (B) Day 5 culture sizes in glutamine-free media with twice the normal glucose concentration (2X GU, y-axis) vs. glutamine-free media with normal glucose levels (x-axis), each normalized to culture in control media. (C-F) Glutamine deprivation responses in the non-tumorigenic exemplar 184A1 and a similarly-sensitive tumorigenic line H3153 (arrows, panel A). (C) Growth curves derived from Cell Titer Glow/ATP content analysis. Numbers below the x-axis, ratio of cell number derived from manual counting (trypan blue) / ATP values. (D) Comparison of AMPK activating phosphorylation, PARP cleavage, and ACC inhibitory phosphorylation. (E) Cell cycle distribution, mitotic figure counts, S-phase fractions, and culture sizes at day 5 culture in indicated conditions. CF, confluent cells in complete media. (F) Day 5 culture sizes differences for high density vs. low density cultures in glutamine-free media. (G) Glutamine consumption (from Figure 1D) vs. glutamine-free culture sizes from part A. (H) hGAC GeneChip hybridization signal vs. glutamine-free culture sizes from part A. Green squares on y-axis are the average hGAC hybridization signals from 3 CD10+, and 3 BerEP4+ purified normal breast samples. See also Figure S2.
Figure 3
Figure 3. Glutamine Restriction Induces S-phase Stalling in a Subset of Basal TNBC
Icon codes in figure keys. Icons represent mean values +/−SD. (A) Growth curves of “Glutamine Sensitive” carcinomas (underlined in Figure 2A) in glutamine-free media. (B) Percent increases in Annexin V reactivity of cells in glutamine-free media at day 3. Group averages; A, 0.8%,+/−1.4; B 1.2% +/−1.4; C, 5.8% +/−3.2; t-test A vs C p= 0.005. (C) Paired bars representing the change in percent of cells in G2/M (Δ% G2/M) with nocodazole treatment at day 5 in control (gray) versus glutamine-free (yellow) media, using cell cycle curve fitting software (FLOJO, Treestar). (D) Paired bars representing the percent S-phase fraction with nocodazole treatment of day 5 cultures in control (gray) vs. glutamine-free (yellow) media. (E) S phase stalling is accompanied by a decrease in total and Serine 780 phosphorylated retinoblastoma protein. (F) Culture confluence reduces S-phase fractions and glutamine sensitivity; S-phase decrease (Δ%), is the decrease in % S-phase with high density culture (gray icons) versus low density culture (colored icons); paired icons connected by dashed lines represent a single cell line. (G) Doubling time (from Figure 1C) vs. glutamine-free culture sizes (from Figure 2H). See also Figure S3.
Figure 4
Figure 4. Common Regulators of Glutamine Metabolism do not Identify Auxotrophic Cells
Icon codes in figure keys; green squares, average hybridization signals from 3 CD10+, and 3 BerEP4+ purified normal breast samples. Icons represent mean values+/−SD. (A) hGAC GeneChip hybridization signal (log2) vs. glutamine-free culture sizes (from Figure 2H) coded as restriction Groups B and C vs. others. (B) GLUL GeneChip hybridization signal (log2) vs. day 5 glutamine-free culture sizes, coded by molecular subtype, dotted lines bracket proliferating, non-tumorigenic sample values. (C) GLUL expression by basal vs. luminal or ER+ vs. ER- samples in 8 clinical breast tumor expression datasets, downloaded from NCBI GEO and Chin 2006 (Chin et al., 2006); t-test p-values below paired boxplots. (D) Correlation of glutamine-free culture sizes and GLUL expression as in panel B, but coded by restriction groups. (E, F) Comparison of (E) GLUL and (F) hGAC mRNA levels at day 3 in glutamine replete versus deficient media, from quantitative PCR analysis; L, low density; C, confluent; Q, glutamine. See also Figure S4 and Tables S4, S5.
Figure 5
Figure 5. Glutamine Auxotrophy Presents Therapeutic Opportunities
Icons represent mean values+/−SD. (A) GeneChip hybridization signals for 4 glutamine transporters and the common heavy chain (SLC3A2) in glutamine auxotrophic cells. (B) GeneChip hybridization signals for transporters as in panel A, for basal carcinoma subsets of clinical datasets downloaded from NCBI GEO and Chin 2006 (Chin et al., 2006), 1 example dataset shown. (C) Relative Asparaginase (y-axis), and DON (x-axis) sensitivities (IC85) of our cell panel, coded by restriction Groups B and C vs. others; dotted line, Asparaginase concentration used to kill sensitive leukemia cells in vitro (1u/ml). (D) Day 5 glutamine-free culture sizes (from Figure 2A) vs. Asparaginase sensitivity (IC50 shown). (E) Four fold drug titrations and calculated IC50s for an exemplar Group C auxotroph (M436). D0, highest drug concentration and calculated IC50s in figure key; Asp, Asparaginase; Pac, Paclitaxel; Dox, Doxorubicin. (F) TCA cycle diagram illustrating respiratory use of glutamine in red arrows. Numbers indicate mass spectroscopy determination of the percent of each metabolite that contains (all / several / no) 13C-carbons derived from culture with 13C-5-glutamine in M436. (G) Decrease in key TCA cycle metabolite pools with glutamine restriction, expressed as % control media cultures; Q-, glutamine-free media. (H) Proliferative effects of siRNA-mediated hGAC mRNA reduction in exemplar cell lines, expressed as a percent of transfection with a scrambled siRNA. See also Figure S5, Tables S6, S7.
Figure 6
Figure 6. Glutamine Restriction and xCT Inhibition Increase ROS
Icon codes in figure keys; NAC, N-acetylcystine; SASP, Sulfasalazine. Icons represent mean values+/−SD. Amino acid quantitation by HPLC. (A) Change in media cystine (xaxis) and glutamate (y-axis) concentrations of cells cultured 24 hours in control media. (B) 24 hrs of glutamine restriction or SASP treatment reduces cystine / glutamate exchange by exemplar Group C auxotrophs; cys, cystine; glu, glutamate. (C) Effects of 24 hour glutamine restriction or SASP treatment on GSH content in an exemplar Group C TNBC; Q-, glutamine-free media; SASP, SASP treatment in complete media; SASP + 2me, SASP treatment in the presence of beta mercaptoethanol. (D) ROS levels in basal carcinomas assessed by DCFHDA fluorescence, normalized to control media reactivity. Light blue, 2 day cultures in glutamine-free media; Group averages; A, 112% +/− 21; B, 121% +/−31; C,162% +/−59; t-test group A vs. C p=0.07; Group A vs. B+C p= 0.074. Light gray, glutamine-free media +NAC. Teal, cultures treated 24 hours with SASP; Group averages; A, 205% +/− 43; B, 287% +/− 62; C, 405% +/− 168; t-test Group A vs. C p= 0.019; Group A vs. B+C p= 0.003. Dark gray, SASP +NAC. (E) Heatmap of genes involved in xCT function; red, increased; green, decreased; N, purified normal CD10+ and BerEp4+ breast epithelial cells; I, proliferating non-tumorigenic cell lines; PE, ER+ tumor cells purified from pleural effusions. (F) SLC7A11 GeneChip hybridization signal (log2) vs. cystine consumption, icons coded by molecular subtype. (G) ROS levels in M231, 48 hours after targeting SLC7A11 or a scrambled siRNA, in the presence or absence of N-acetylcystine (NAC). (H) SLC7A11 knockdown efficiency of siRNAs used in part G. See also Figure S6.
Figure 7
Figure 7. SASP Attenuates Proliferation In vitro and In vivo
Icon codes in figure keys. Icons represent mean values+/−SD. (A) Cystine consumption in complete media derived from HPLC analysis (x-axis) versus SASP sensitivity (IC50). (B) NAC treatment rescues SASP-induced culture size defects in an exemplar cell from each restriction group A-C. (C) SASP treatment attenuates xenograft growth; Group average tumor volumes separation p-values noted on graph. (D) Examples of xCT expression in exemplar human TNBC tumor sections; nuclei, blue; xCT-specific HRP signal, brown Upper positive, lower negative. (E) SASP reduces the Carboplatin IC50 of most basal TNBC; paired icons connected by dashed lines represent a single TNBC; colored icons, Carboplatin IC50; gray icons, IC50 of Carboplatin plus 300 µM SASP; Q insens., basal TNBC with less glutamine-sensitivity than non-tumorigenic cells. (F) Summary of discussed glutamine catabolic activities. Red, compounds tested in this manuscript; green, activities with potential therapeutic inhibitory importance in glutamineavid TNBC; dark blue, LAT1 glutamine/leucine antiporter and ASCT2, system ASC glutamine transporter; gray, xCT, the glutamate/cystine antiporter; light blue, glutamine anaplerosis path; SASP, Sulfasalazine; ASP, Asparaginase; DON, 6-diazo-5-oxo-Lnorleucine; ATs, various aminotransferases; GS, glutamine synthase; GLS, glutaminase; hGAC, carboxy-terminal splice variant of glutaminase. Not all uses of intracellular glutamine or DON targets are illustrated. See also Figure S7.

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