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Front Pharmacol
2018 Jul 19;9:785. doi: 10.3389/fphar.2018.00785.
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Disruption of Amino Acid Homeostasis by Novel ASCT2 Inhibitors Involves Multiple Targets.
Bröer A
,
Fairweather S
,
Bröer S
.
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The glutamine transporter ASCT2 (SLC1A5) is actively investigated as an oncological target, but the field lacks efficient ASCT2 inhibitors. A new group of ASCT2 inhibitors, 2-amino-4-bis(aryloxybenzyl)aminobutanoic acids (AABA), were developed recently and shown to suppress tumor growth in preclinical in vivo models. To test its specificity, we deleted ASCT2 in two human cancer cell lines. Surprisingly, growth of parental and ASCT2-knockout cells was equally sensitive to AABA compounds. AABA compounds inhibited glutamine transport in cells lacking ASCT2, but not in parental cells. Deletion of ASCT2 and amino acid (AA) depletion induced expression of SNAT2 (SLC38A2), the activity of which was inhibited by AABA compounds. They also potently inhibited isoleucine uptake via LAT1 (SLC7A5), a transporter that is upregulated in cancer cells together with ASCT2. Inhibition of SNAT2 and LAT1 was confirmed by recombinant expression in Xenopus laevis oocytes. The reported reduction of tumor growth in pre-clinical models may be explained by a significant disruption of AA homeostasis.
FIGURE 1. Inhibition of tumor cell growth by AABA. Structure of 2-amino-4-bis(aryloxybenzyl)aminobutanoic acids compound 12 (A) and V-9302 (B) as described by Schulte et al. (2016, 2018). (C) The presence of ASCT2 in wild-type and genome-edited 143B cells was evaluated by western blotting of cell homogenates using an ASCT2-specific antibody. (D) Growth of parental and ASCT2ko 143B cells was monitored using IncuCyte technology in the presence of increasing concentrations of compound 12 (n = 12, cells were seeded from at least three different batches). (E) Reproducibility of cell growth assays in a well-to-well comparison showing growth in DMEM/F12 supplemented with 2-mM glutamine and BME supplemented with 0.5-mM glutamine. Concentration of compound 12 is indicated in the margin. (F) Growth of parental and ASCT2ko 143B cells was monitored using IncuCyte technology in the presence of increasing concentrations of V-9302 (n = 10, cells were seeded from at least three different batches).
FIGURE 2. Inhibition of glutamine transport by AABA in wild-type and ASCT2ko cells. (A) Uptake of [14C]glutamine (100 μM) was measured in 143B cells [parental (wt) and ASCT2ko] in the presence or absence of 100 μM compound 12. Before transport, cells were kept in DMEM/F12 medium supplemented with 10% FCS (+AA) or in Hank’s buffered salts solution supplemented with 5-mM glucose and 1% dialyzed FCS (–AA) for 12 h [parental (wt) n = 5 and ASCT2ko n = 9 for each condition]. p-Values are indicated in the figure. (B) 143B cells and HCC1806 cells were incubated as in A and used for surface biotinylation and subsequent detection of glutamine transporters by immunoblotting. Na+, K+-ATPase was used as a loading control; the SNAT2-specific band is indicated by an asterisk (*) (n = 3). (C) Detection of ASCT2 in cell homogenates of parental and genome edited HCC1806 triple-negative breast cancer cells. Actin was used as a loading control (n = 5). (D) Uptake of [14C]glutamine (100 μM) was measured in HCC1806 cells [parental (wt) and ASCT2ko] in the presence or absence of 100 μM compound 12. Before transport, cells were kept in DMEM/F12 medium supplemented with 10% FCS (+AA) or in Hank’s buffered salts solution supplemented with 5-mM glucose and 1% dialyzed FCS (–AA) for 12 h (parental n = 5 and ASCT2ko n = 5 for each condition). p-Values are indicated in the figure.
FIGURE 3. Inhibition of system A and LAT1 activity by AABA. (A) Uptake of [14C]glutamine (100 μM) was measured in parental (wt) or ASCT2ko 143B cells in the presence (n = 9 each group) or absence of 100 μM compound 12 (n = 9 each group) or 10-mM MeAIB (n = 6). Before transport, cells were kept in DMEM/F12 medium supplemented with 10% FCS (+AA) or in Hank’s buffered salts solution plus 5-mM glucose and 1% dialyzed FCS (–AA) for 12 h. n.s. difference not significant. p-Values for significant differences are shown in the figure. (B) Uptake of [14C]isoleucine (100 μM) was measured in Na+-free buffer in 143B cells (n = 6), in the presence (n = 6) or absence (n = 6) of 100 μM compound 12 or 2 μM JPH203 (n = 6) n.s. difference not significant. (C) Uptake of [14C]glutamine was measured as in A, but V-9302 was used instead of compound 12 (n = 3, p-values indicated in the figure). (D) Uptake of [14C]isoleucine was measured as in B, but V-9302 was used instead of compound 12 (n = 3, p-values indicated in the figure).
FIGURE 4. Evaluation of inhibitor specificity. (A) Transporters hSNAT1, hSNAT2, hASCT2, and h4F2hc/rLAT1 were expressed in Xenopus laevis oocytes. Transport experiments were performed 4–7 days after injection of the corresponding cRNAs. Glutamine uptake was used to measure hSNAT1, hSNAT2, and hASCT2 activity, while isoleucine transport was used to measure h4F2hc/rLAT1 activity. Uptake was measured in the presence or absence of inhibitors as indicated (n = 8 for each transporter) in three batches of oocytes. (B) A homology model of human LAT1 was generated after alignment with the E. coli arginine–agmatine antiporter (PDB 5J4I). Docking of V-9302 was performed using PyRX. One of several energetically favorable poses is shown. (C) Delineation of glutamine transport pathways in 143B osteosarcoma cells. The contributions of SNAT1, SNAT2, ASCT2, and LAT1 were deduced using combinations of inhibitors (n = 6). Groups that were different (p < 0.001) have different letter labeling.
FIGURE 5. A model of amino acid homeostasis in cancer cells. The physiological role of SNAT1 is to accumulate non-essential amino acids in the cytosol (loader). These are used as exchange substrates to bring the remaining neutral amino acids into the cytosol via exchangers ASCT2 and LAT1, thus harmonizing amino acid pools. Blockade of LAT1 causes a rescue response resulting in the induction of SNAT2. Block of these two transporters impairs accumulation and harmonization of amino acid pools in cancer cells and also negates the rescue response.
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