TY - JOUR
AU1 - Furusawa, Akiko
AU2 - Miyamoto, Morikazu
AU3 - Takano, Masashi
AU4 - Tsuda, Hitoshi
AU5 - Song, Yong Sang
AU6 - Aoki, Daisuke
AU7 - Miyasaka, Naoyuki
AU8 - Inazawa, Johji
AU9 - Inoue, Jun
AB - Abstract Amino acids (AAs) are biologically important nutrient compounds necessary for the survival of any cell. Of the 20 AAs, cancer cells depend on the uptake of several extracellular AAs for survival. However, which extracellular AA is indispensable for the survival of cancer cells and the molecular mechanism involved have not been fully defined. In this study, we found that the reduction of cell survival caused by glutamine (Gln) depletion is inversely correlated with the expression level of glutamine synthetase (GS) in ovarian cancer (OVC) cells. GS expression was downregulated in 45 of 316 OVC cases (14.2%). The depletion of extracellular Gln by treatment with l-asparaginase, in addition to inhibiting Gln uptake via the knockdown of a Gln transporter, led to the inhibition of cell growth in OVC cells with low expression of GS (GSlow-OVC cells). Furthermore, the re-expression of GS in GSlow-OVC cells induced the inhibition of tumor growth in vitro and in vivo. Thus, these findings provide novel insight into the development of an OVC therapy based on the requirement of Gln. Introduction Ovarian cancer (OVC) is the fifth cause of cancer-related death in women. Epithelial ovarian cancer comprises 90% of all OVCs and is classified into four histologic subtypes of serous, mucinous, endometrioid and clear cell carcinomas (1,2). OVC is a global problem, is typically diagnosed at a late stage and is frequently relapse (3). Therefore, it is necessary to identify novel and efficient treatment for human epithelial ovarian cancer. Amino acids (AAs) are biologically important nutrient compounds necessary for the survival of any cell. Of the 20 AAs, 9 essential amino acids (EAAs) cannot be synthesized endogenously and must be obtained as extracellular components. In contrast, 11 nonessential amino acids (NEAAs) can be endogenously synthesized by each corresponding enzyme (4–6). Accumulating evidence has demonstrated that certain types of cancer cells depend on several NEAAs, including arginine, glycine, leucine, glutamine (Gln), proline and serine, for cell survival (7–13). However, which extracellular AA is indispensable for the survival of cancer cells and the underlying molecular mechanism have not been fully defined. On the other hand, it is known that l-asparaginase (l-asp), which is one of the most important drugs used for acute lymphoblastic leukemia (ALL) therapy, is an enzyme that catalyzes the hydrolysis of asparagine or Gln to aspartate or glutamate (Glu), respectively. The l-asp-mediated depletion of blood asparagine is the basic therapeutic concept in ALL because ALL cells cannot synthesize endogenous asparagine due to the lack of asparagine synthetase (ASNS), resulting in the dependency on extracellular asparagine uptake (14,15). Additionally, argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis and prolonged arginine starvation by exposure to pegylated arginine deiminase (ADI-PEG20) sensitive in ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs, and depend on extracellular arginine uptake (11). Thus, the absence of a biosynthetic enzyme for any of the NEAAs leads to the insufficient intracellular production of that NEAA in the tumor of each individual cancer patient. Therefore, the depletion of specific AAs is expected to be therapeutically useful for precision cancer medicine. In this study, we found that the downregulation of glutamine synthetase (GS) expression led to the increased sensitivity to Gln depletion in OVC cells. GS expression was downregulated in a subset of primary OVC cases. The depletion of extracellular Gln by treatment with l-asp, in addition to inhibiting Gln uptake via the knockdown of a Gln transporter, led to the inhibition of cell growth in OVC cells with low expression of GS (GSlow-OVC cells). These findings provide novel insight into the development of OVC therapy based on the requirement of Gln. Materials and methods Cell lines and cell culture A total of 14 OVC cell lines were used in this study. The A2780, KK, KF28, MH, OVCAR-3, OVCAR-5, OVCAR-8 and SNU119 cells were maintained in RPMI 1640 medium (WAKO, Osaka, Japan); A#39 cells were maintained in DMEM/F12 medium (WAKO); CaOV3 cells were maintained in DMEM medium (WAKO); ES-2 and SK-OV-3 cells were maintained in McCoy’s 5a medium (Thermo Fisher Scientific, Waltham, MA); the TOV21G cells were maintained in a 1:1 mixture of MCDB-105/M199 medium (Cell Applications, San Diego, CA/Thermo Fisher Scientific) and the RMG-1 cells were maintained in Ham’s F12 medium (WAKO). Each culture medium was supplemented with 10% fetal bovine serum. The cultures were maintained at 37°C with 5% CO2. Of the 14 OVC cell lines used, ES-2, OVCAR-3 and SK-OV-3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA); KK, KF28 and MH were from the National Defense Medical College (Saitama, Japan) (16); A2780, SNU119, A#39, CaOV3 and TOV21G were kindly provided by Dr Y.S.S. (Seoul National University, Seoul, Korea); RMG-1 cell line was kindly provided by Dr D.A. (Keio University School of Medicine, Tokyo, Japan) (17). Once resuscitated, the cell lines were authenticated through monitoring of cell morphology. Patients and tumor specimens Formalin-fixed, paraffin-embedded tissue blocks of primary epithelial OVCs from 316 patients were used to construct tissue microarrays. All patients underwent surgery at the National Defense Medical College (NDMC) Hospital (Saitama, Japan) from 1986 to 2006 with a 10 year follow-up period starting from the initial surgery. The median follow-up period was 59.5 months (range, 1–257 months). Of the 316 patients, 114 (36.1%) died due to their cancer. The tumor histological types were classified according to the WHO criteria. The clinical stages of the disease were classified according to the 1988 International Federation of Gynecology and Obstetrics (FIGO) system. All patients provided written informed consent in a formal style before the study. This study was approved by the ethics committees of the NDMC and Tokyo Medical and Dental University (TMDU). Antibodies and reagents Antibodies for GS (G2781), ASNS (HPA029318), LC3B (L7543) and β-actin (A5441) were purchased from Sigma–Aldrich (St. Louis, MO). Antibodies directed against ASCT2 (#5100) were from Cell Signaling (Danvers, MA). l-Asparaginase (l-asp; LEUNASE) was purchased from Kyowa Hakko Kirin Co (Tokyo, Japan). Bafilomycin A1 (Baf.A1) was purchased from Sigma–Aldrich. Preparation of single AA-depleted medium Conditioned medium, a single AA-depleted medium, was prepared by adding the other 19 AAs (at a final concentration of 100 µM), excluding one AA, to AA-free RPMI 1640 medium (WAKO) supplemented with 0.1% fetal bovine serum. Alanine, aspartate, Glu, glycine, leucine, methionine, serine, tryptophan, tyrosine and valine were purchased from WAKO. Arginine, asparagine, cysteine, Gln, histidine, isoleucine, lysine, phenylalanine, proline and threonine were purchased from Peptide Institute (Osaka, Japan). Transfection of siRNAs Small-interfering RNAs (siRNAs) for GS (siGENOME SMARTpool; M-008228-01-0005), ASCT2 (siGENOME SMARTpool; M-007429-01-0005), a Gln transporter and a nontargeting negative control (siGENOME SMARTpool; D-001206-14-05) were obtained from Thermo Fisher Scientific. The cells were transfected with each siRNA at 20 nM using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Plasmid construction The PCR product containing the coding region of GS was inserted into a pCMV-3Tag-1A (Flag tagged) expression vector (Stratagene, San Diego, CA). Short hairpin RNA (shRNA) oligonucleotides for GS gene (target sequence: 5′-CCCTAAACCTCTACCCTAC-3′) were annealed and inserted into the pGreenPuro vector (System Biosciences, Mountain View, CA). Cell survival assay Cells were seeded and cultured in conditioned medium or were treated for 48 h. Cell survival was assessed using a crystal violet staining assay. The cells were washed in phosphate-buffered saline (PBS) and were fixed with 0.1% crystal violet in 10% formaldehyde in PBS for 5 min. The excess crystal violet solution was discarded, and after air-drying completely, the stained cells were lysed with a 2% SDS solution by shaking the plates for 1 h. The optical density was measured at 560 nm using a microplate reader. The percentage absorbance was calculated for each well. The optical density values of cells in the control wells were arbitrarily set to 100% to determine the percentage of viable cells. Western blot analysis Whole-cell lysates were subjected to SDS-PAGE, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare UK Ltd, Little Chalfont, UK). After blocking with TBS containing 0.05% Tween-20 and 5% non-fat dry milk for 1 h, the membranes were incubated with primary antibodies overnight. The dilutions for the primary antibodies were 1/2000 for rabbit anti-GS antibody, 1/2000 for anti-ASNS antibody, 1/5000 for anti-LC3B antibody, 1/1000 for anti-ASCT2 antibody and 1/5000 for mouse anti-β-actin antibody. The membranes were washed and exposed to horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) antibodies (both at 1/5000) for 1 h. The bound antibodies were visualized with a horseradish peroxidase staining solution or with an ECL Western detection kit according to the manufacturer’s instructions (Cell Signaling Technology). Generation of GS-expressing cells and GS-inhibited cells Flag-tagged empty vector (Vec) or GS vector was transfected into OVC cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, and the transfected cells were then selected by treatment with G418 (Sigma–Aldrich). Lentivirus for the transduction of the shRNA vectors was prepared using the pPACK Packaging Kit (System Biosciences, Palo Alto, CA) according to the manufacturer’s instructions. The virus titer was measured in infectious units/ml via an RT-PCR-based method using the Global UltraRapid Lentiviral Titer Kit (System Biosciences). KF28 cells were infected with five multiplicity of infection (MOI) of lentivirus with either empty vector (as a control) or the GS-shRNA vector using TransDux (System Biosciences). The selection of infected cells was performed by treatment with 1 µg/ml puromycin (Sigma–Aldrich). Determination of the sub-G1 cell population by fluorescence-activated cell sorting analysis Both attached cells and cells floating in the medium were collected, washed in PBS and fixed with 70% cold ethanol on ice for 30 min. Fixed cells were washed in PBS again, and incubated in PBS containing RNase (100 µg/ml) for 30 min at 37°C. Cells were then stained with propidium iodide (Invitrogen), and cell population analysis was performed using the AccuriⓇ Flow Cytometer. Three-dimensional spheroid culture Ninety-six-well plates were precoated with Matrigel as a basement membrane by adding 75 μl of Matrigel (BD Biosciences, San Jose, CA) to each well and incubating at 37°C for 30 min to allow the Matrigel to solidify. Cells (3.0 × 103 per well) that were suspended in standard medium containing 2% Matrigel were then seeded onto the Matrigel-precoated wells. All microscopy images were captured using a Nikon Eclipse E400 (Nikon, Tokyo, Japan), and the spheroid size was measured using ImageJ software (National Institutes of Health, MD). The size relative to the size on the third day was calculated. In vivo tumor growth assay Seven-week-old female BALB/c nude mice were purchased from the Charles River Laboratories Japan (Yokohama, Japan). A laparotomy with a transverse incision was performed under anesthesia. Cells (1 × 107 cells in 100 µl of PBS with 50% Matrigel) were injected subcutaneously into the flank of the nude mice. ES-2 cells (1 × 107 cells in 200 µl of PBS) were injected into the intraperitoneal cavity of the nude mice. Immunohistochemical analysis We constructed tissue microarrays from tissue blocks prepared from 316 OVC tumors using a Tissue Microarrayer (Beecher Instruments, Silver Spring, MD) as previously described (18). Immunohistochemistry was performed on tissue microarray sections. The sections were deparaffinized in xylene and were rehydrated using a graded ethanol series (100, 90, 80, 70 and 50%) to water. After the retrieval of antigens by boiling in 10 mM citrate buffer (pH 6.0), the sections were treated with 0.3% hydrogen peroxide in methanol to inactivate endogenous peroxidase. Nonspecific binding was blocked by incubating in goat serum in PBS. Next, the slides were incubated with rabbit anti-GS antibodies (dilution for both: 1/2000) overnight at room temperature. The bound antibody was visualized using diaminobenzidine as a chromogen (VECTASTAIN Elite ABC kit, Vector Laboratories, Burlingame, CA), and the sections were lightly counterstained with hematoxylin. The immunohistochemical evaluation was performed by three researchers (A.F., Ju.I. and H.T.) according to our previous studies (18). Briefly, the intensity score for cytoplasmic GS expression was determined using the Histo-score (H-score), which was calculated by the semiquantitative assessment of both the intensity of staining (graded as follows: 0, nonstaining; 1+, weak; 2+, medium; 3+, strong) and the percentage of positive cells. The formula was (3 × the percentage of cells positive for 3+ staining) + (2 × the percentage of cells positive for 2+ staining) + (1 × the percentage of cells positive for 1+ staining), yielding a range of 0–300 (19). The expression level of each component was categorized as low or high according to the median H-score value. Measurement of the Gln level in medium The Gln level in the medium was measured using the Glutamine Assay Kit (ab19701, Abcam, Cambridge, UK). Statistical analysis The correlation between GS expression in primary OVCs and the clinicopathological variables were analyzed using the chi-squared test or Fisher’s exact test. For in vitro assays, differences in the population mean between the control and test groups were compared using Student’s t-test. Differences with P values lower than 0.05 were considered to be statistically significant. Results Differential AA requirements for cell survival in a panel of 14 OVC cell lines To determine the requirement of each AA for cell survival in 14 OVC cell lines, we examined the sensitivity to culturing in conditioned medium in which the corresponding single AA was depleted (Figure 1A). Since EAAs cannot be endogenously synthesized due to the absence of each of the corresponding biosynthetic enzymes, the uptake of extracellular EAAs is critical for cell survival (3–6,20). Nevertheless, in 9 (KK, OVCAR-5, OVCAR-8, SK-OV-3, RMG-1, A#39, SNU119, CaOV3 and MH) of the 14 OVC cell lines, the depletion of any EAA did not affect cell survival, suggesting that these cell lines may be metabolically reprogrammed to survive even under an EAA-depleted condition. However, while NEAAs can be endogenously produced by biosynthetic enzymes (7), we found that the depletion of some NEAAs, either arginine, asparagine, Gln, glycine, proline, serine or tyrosine, had an effect on cell survival in 10 of the OVC cell lines but not the A#39, SNU119, CaOV3 or MH cells. This result suggests that the affected OVC cells may depend on the uptake of the respective extracellular NEAA for cell survival due to the lack of a corresponding synthetase. Therefore, these findings suggest that there may be a cell-type-dependent AA requirement for cell survival in OVC cells. Effect of GS expression on cell survival under Gln-depleted conditions While it is believed that almost all cancer cells depend on Gln for cell survival or proliferation, the so-called Gln addiction (21), we found that Gln depletion did not affect 8 of the 14 OVC cell lines (Figure 1A). Therefore, we hypothesized that the expression of GS, which catalyzes the production of endogenous Gln from Glu, may be involved in the sensitivity to Gln depletion (Figure 1B). As a result, we showed a positive correlation between the cell survival rate under a condition of Gln depletion and the expression level of the GS protein in 14 cell lines (P = 0.048, R2 = 0.28; Figure 1C and D), suggesting that a decrease in the GS protein level may lead to OVC cell dependence on extracellular Gln. Indeed, when FLAG-tagged GS protein was stably expressed in the GSlow-OVC cells, OVCAR-3 or OVCAR-8 cells, the cell survival rate of each cell line under Gln depletion was remarkably increased (Figure 1E). By contrast, shRNA- or siRNA-mediated inhibition of GS expression in the GShigh-OVC cells, KF28 cells, resulted in a decrease in the cell survival rate under conditions of Gln depletion (Figure 1F). In addition, the inhibition of Gln uptake by the knockdown of ASCT2, a Gln transporter, was effective in the GSlow-OVC cells (P = 0.01 for OVCAR-3 cells) but not in the GShigh-OVC cells (P = 0.29 for KF28 cells; Figure 1G). Taken together, these results suggest that GS expression is critical regarding the requirement of extracellular Gln for OVC cell survival. Figure 1. View largeDownload slide Effect of GS expression on cell survival under Gln-depleted conditions. (A) Differential AA requirements for cell survival in 14 OVC cell lines. Gray boxes indicate more than 30% reduction in the cell survival rate for each OVC cell line cultured in each single AA-depleted medium compared with that of culture in the standard medium. (B) GS- and glutaminase (GLS)-catalyzed reactions. (C) Western blot analysis of GS in the 14 OVC cell lines. Cell lysates were subjected to SDS-PAGE and were incubated with the indicated antibodies. (D) Positive correlation between the expression level of the GS protein and the cell survival rate under Gln-depleted conditions in the 14 OVC cell lines. (E) Effect of GS overexpression on the cell survival rate under Gln-depleted conditions. Flag-tagged empty vector (Vec) or GS protein (GS) was stably expressed in low-GS-expressing OVCAR-3 or OVCAR-8 cells. Upper panels: western blot analysis of the GS protein. Lower panels: cell survival rates under Gln-depleted conditions are indicated. Bar, SD. *P < 0.05 and **P < 0.01. (F) Effect of GS knockdown on the cell survival rate under Gln-depleted conditions. GS expression was inhibited by shRNA or siRNA in the negative control (NC) or GS in the high-GS-expressing KF28 cells. Upper and Lower panels are presented as described in (E). *P < 0.05 and **P < 0.01. (G) Effect of ASCT2 knockdown on the cell survival rate under the standard condition. ASCT2 expression was inhibited by siRNA for the negative control (NC) or ASCT2 in the low-GS-expressing OVCAR-3 (left panel) or high-GS-expressing KF28 cells (right panel). The Upper and Lower panels are presented as described in (E). **P < 0.05. Figure 1. View largeDownload slide Effect of GS expression on cell survival under Gln-depleted conditions. (A) Differential AA requirements for cell survival in 14 OVC cell lines. Gray boxes indicate more than 30% reduction in the cell survival rate for each OVC cell line cultured in each single AA-depleted medium compared with that of culture in the standard medium. (B) GS- and glutaminase (GLS)-catalyzed reactions. (C) Western blot analysis of GS in the 14 OVC cell lines. Cell lysates were subjected to SDS-PAGE and were incubated with the indicated antibodies. (D) Positive correlation between the expression level of the GS protein and the cell survival rate under Gln-depleted conditions in the 14 OVC cell lines. (E) Effect of GS overexpression on the cell survival rate under Gln-depleted conditions. Flag-tagged empty vector (Vec) or GS protein (GS) was stably expressed in low-GS-expressing OVCAR-3 or OVCAR-8 cells. Upper panels: western blot analysis of the GS protein. Lower panels: cell survival rates under Gln-depleted conditions are indicated. Bar, SD. *P < 0.05 and **P < 0.01. (F) Effect of GS knockdown on the cell survival rate under Gln-depleted conditions. GS expression was inhibited by shRNA or siRNA in the negative control (NC) or GS in the high-GS-expressing KF28 cells. Upper and Lower panels are presented as described in (E). *P < 0.05 and **P < 0.01. (G) Effect of ASCT2 knockdown on the cell survival rate under the standard condition. ASCT2 expression was inhibited by siRNA for the negative control (NC) or ASCT2 in the low-GS-expressing OVCAR-3 (left panel) or high-GS-expressing KF28 cells (right panel). The Upper and Lower panels are presented as described in (E). **P < 0.05. Downregulation of GS expression in primary OVC samples We examined the expression status of the GS protein via immunohistochemical analysis in 316 primary OVC samples. The expression of the GS protein was assessed using the H-score based on the staining intensity and percentage of positive cells (see the Materials and methods). The H-score in normal fimbria of the fallopian tube was 200, and cases with an H-score less than 100 were defined as having ‘downregulation,’ as shown by the immunostaining of serous carcinoma for case 1 (Figure 2A). We showed that the expression of the GS protein was downregulated in 12 of 131 serous carcinoma cases (9.2%; Figure 2B). The downregulation of GS was shown in 28 of 87 cases (32.1%) of clear cell carcinoma, in 3 of 44 cases (6.8%) of endometrioid carcinoma, and in 2 of 54 cases (3.7%) of mucinous carcinoma (Table 1). In total, GS expression was downregulated in 45 of all 316 cases of OVC tumors (14.2%). However, there was no significant association between any clinical implications and GS expression status. Figure 2. View largeDownload slide Immunostaining of the GS protein in primary ovarian high-grade serous carcinoma (HGSC). (A) Representative images for normal fimbria and two cases (case 1 and case 2) of primary HGSC. Squares in the upper panels are enlarged in the lower panels. The H-score for normal fimbria (left), case 1 (middle) or case 2 (right) was 200, 0 or 300, respectively. Bar: 100 µm (upper panels), 10 µm (lower panels). (B) The expression status of GS in primary HGSC (n = 131). The H-score was calculated as described in the Materials and methods section. Cases with an H-score less than 100, as indicated by gray bars, were defined as ‘downregulation’ (12 of 131 cases, 9.2%). Figure 2. View largeDownload slide Immunostaining of the GS protein in primary ovarian high-grade serous carcinoma (HGSC). (A) Representative images for normal fimbria and two cases (case 1 and case 2) of primary HGSC. Squares in the upper panels are enlarged in the lower panels. The H-score for normal fimbria (left), case 1 (middle) or case 2 (right) was 200, 0 or 300, respectively. Bar: 100 µm (upper panels), 10 µm (lower panels). (B) The expression status of GS in primary HGSC (n = 131). The H-score was calculated as described in the Materials and methods section. Cases with an H-score less than 100, as indicated by gray bars, were defined as ‘downregulation’ (12 of 131 cases, 9.2%). Table 1. Correlation between GS expression and clinicopathological variables in 316 patients with epithelial OVC H-score All 0 1–99 100–199 200–300 n n % n % n % n % 316 10 3.2 54 17.1 160 50.6 92 29.1 Age <50 118 4 3.4 9 7.6 34 28.8 71 60.2 ≥50 198 6 3.0 26 13.1 61 30.8 105 53.0 Histological type Serous carcinoma 131 3 2.3 9 6.9 43 32.8 76 58.0 Clear cell carcinoma 87 7 8.0 21 24.1 32 36.8 27 31.0 Endometrioid carcinoma 44 0 0.0 3 6.8 13 29.5 28 63.6 Mucinous carcinoma 54 0 0.0 2 3.7 7 13.0 45 83.3 FIGO stage I 122 4 3.3 15 12.3 30 24.6 73 59.8 II 38 1 2.6 6 15.8 14 36.8 17 44.7 III 117 4 3.4 11 9.4 39 33.3 63 53.8 IV 38 1 2.6 4 10.5 10 26.3 23 60.5 Recurrence Yes 155 4 2.6 17 11.0 49 31.6 85 54.8 No 161 6 3.7 18 11.2 46 28.6 91 56.5 H-score All 0 1–99 100–199 200–300 n n % n % n % n % 316 10 3.2 54 17.1 160 50.6 92 29.1 Age <50 118 4 3.4 9 7.6 34 28.8 71 60.2 ≥50 198 6 3.0 26 13.1 61 30.8 105 53.0 Histological type Serous carcinoma 131 3 2.3 9 6.9 43 32.8 76 58.0 Clear cell carcinoma 87 7 8.0 21 24.1 32 36.8 27 31.0 Endometrioid carcinoma 44 0 0.0 3 6.8 13 29.5 28 63.6 Mucinous carcinoma 54 0 0.0 2 3.7 7 13.0 45 83.3 FIGO stage I 122 4 3.3 15 12.3 30 24.6 73 59.8 II 38 1 2.6 6 15.8 14 36.8 17 44.7 III 117 4 3.4 11 9.4 39 33.3 63 53.8 IV 38 1 2.6 4 10.5 10 26.3 23 60.5 Recurrence Yes 155 4 2.6 17 11.0 49 31.6 85 54.8 No 161 6 3.7 18 11.2 46 28.6 91 56.5 View Large Table 1. Correlation between GS expression and clinicopathological variables in 316 patients with epithelial OVC H-score All 0 1–99 100–199 200–300 n n % n % n % n % 316 10 3.2 54 17.1 160 50.6 92 29.1 Age <50 118 4 3.4 9 7.6 34 28.8 71 60.2 ≥50 198 6 3.0 26 13.1 61 30.8 105 53.0 Histological type Serous carcinoma 131 3 2.3 9 6.9 43 32.8 76 58.0 Clear cell carcinoma 87 7 8.0 21 24.1 32 36.8 27 31.0 Endometrioid carcinoma 44 0 0.0 3 6.8 13 29.5 28 63.6 Mucinous carcinoma 54 0 0.0 2 3.7 7 13.0 45 83.3 FIGO stage I 122 4 3.3 15 12.3 30 24.6 73 59.8 II 38 1 2.6 6 15.8 14 36.8 17 44.7 III 117 4 3.4 11 9.4 39 33.3 63 53.8 IV 38 1 2.6 4 10.5 10 26.3 23 60.5 Recurrence Yes 155 4 2.6 17 11.0 49 31.6 85 54.8 No 161 6 3.7 18 11.2 46 28.6 91 56.5 H-score All 0 1–99 100–199 200–300 n n % n % n % n % 316 10 3.2 54 17.1 160 50.6 92 29.1 Age <50 118 4 3.4 9 7.6 34 28.8 71 60.2 ≥50 198 6 3.0 26 13.1 61 30.8 105 53.0 Histological type Serous carcinoma 131 3 2.3 9 6.9 43 32.8 76 58.0 Clear cell carcinoma 87 7 8.0 21 24.1 32 36.8 27 31.0 Endometrioid carcinoma 44 0 0.0 3 6.8 13 29.5 28 63.6 Mucinous carcinoma 54 0 0.0 2 3.7 7 13.0 45 83.3 FIGO stage I 122 4 3.3 15 12.3 30 24.6 73 59.8 II 38 1 2.6 6 15.8 14 36.8 17 44.7 III 117 4 3.4 11 9.4 39 33.3 63 53.8 IV 38 1 2.6 4 10.5 10 26.3 23 60.5 Recurrence Yes 155 4 2.6 17 11.0 49 31.6 85 54.8 No 161 6 3.7 18 11.2 46 28.6 91 56.5 View Large Effect of GS expression on the sensitivity to treatment with l-asp We hypothesized that the depletion of extracellular Gln may affect cell growth inhibition in OVC cells together with the downregulation of GS expression. l-asp, one of the most important drugs used for ALL therapy, is an enzyme that catalyzes the hydrolysis of asparagine or Gln to aspartate or Glu, respectively (Figure 3A). In the present study, we confirmed that treatment with l-asp induced the complete depletion of Gln in the culture media (Figure 3B). We next examined the cell sensitivity to l-asp treatment in the 14 OVC cell lines and found a positive, but not statistically significant, correlation between the expression level of the GS protein and the cell survival rate with l-asp treatment of the 14 OVC cell lines (P = 0.89, R2 = 0.12; Figure 3C). As shown in Figure 1A, asparagine depletion of the medium did not affect the survival of the OVC cells, suggesting that the l-asp-induced cytotoxicity is attributed to Gln depletion. Furthermore, the cell survival rate with l-asp treatment was significantly increased in the OVCAR-3 or OVCAR-8 cells that stably expressed GS and was decreased in the GS-inhibited KF28 cells compared with that in each of the control–vector cells (Figure 3D). We also showed that apoptotic cell death induced by l-asp treatment was remarkably inhibited in the OVCAR-3 cells that stably expressed GS, compared with that in the control–vector cells (13.4% in control–vector cells, 5.0% in GS-expressing cells, P = 0.009; Supplementary Figure 1, available at Carcinogenesis Online). Figure 3. View largeDownload slide Effect of GS expression on the sensitivity to l-asp treatment. (A) l-asp can catalyze the hydrolysis of asparagine or Gln to aspartate or Glu, respectively. (B) Measurement of the Gln level in the medium. OVCAR-3 cells were cultured in standard medium with or without 1.5 U/ml l-asp. After 24 h, the Gln level in the medium was measured. ND: not detected. (C) Positive correlation between the expression level of the GS protein and the cell survival rate under conditions of l-asp-treatment in the 14 OVC cell lines. Relative cell survival rates with l-asp treatment (1.25 U/ml, 2 days) were measured in 14 OVC cell lines. (D) Cell survival assay with l-asp treatment. Flag-tagged empty vector (Vec) or GS-expressing OVCAR3 or OVCAR8 cells and shRNA-negative control (sh-NC) or GS (sh-GS)-expressing KF28 cells were treated with l-asp at the indicated concentrations for 2 days. The relative cell survival rates are indicated. Bar, SD. *P < 0.05. (E) and (F) Western blot analysis of l-asp-treated OVC cells. Cells were treated with l-asp (10 U/ml) and/or bafilomycin A1 (Baf.A1, 100 nM) for 2 days. Cell lysates were subjected to SDS-PAGE and were then incubated with the indicated antibodies. The arrow indicates the band for LC3B form-II, an autophagosome marker. Figure 3. View largeDownload slide Effect of GS expression on the sensitivity to l-asp treatment. (A) l-asp can catalyze the hydrolysis of asparagine or Gln to aspartate or Glu, respectively. (B) Measurement of the Gln level in the medium. OVCAR-3 cells were cultured in standard medium with or without 1.5 U/ml l-asp. After 24 h, the Gln level in the medium was measured. ND: not detected. (C) Positive correlation between the expression level of the GS protein and the cell survival rate under conditions of l-asp-treatment in the 14 OVC cell lines. Relative cell survival rates with l-asp treatment (1.25 U/ml, 2 days) were measured in 14 OVC cell lines. (D) Cell survival assay with l-asp treatment. Flag-tagged empty vector (Vec) or GS-expressing OVCAR3 or OVCAR8 cells and shRNA-negative control (sh-NC) or GS (sh-GS)-expressing KF28 cells were treated with l-asp at the indicated concentrations for 2 days. The relative cell survival rates are indicated. Bar, SD. *P < 0.05. (E) and (F) Western blot analysis of l-asp-treated OVC cells. Cells were treated with l-asp (10 U/ml) and/or bafilomycin A1 (Baf.A1, 100 nM) for 2 days. Cell lysates were subjected to SDS-PAGE and were then incubated with the indicated antibodies. The arrow indicates the band for LC3B form-II, an autophagosome marker. We previously demonstrated that autophagy serves a cytoprotective function in l-asp-treated ALL cells (22); therefore, we next attempted to define the effect of autophagy activity on the sensitivity of OVC cells to treatment with l-asp. The autophagy flux in three OVC cell lines, two l-asp-sensitive cell lines (OVCAR-3 and OVCAR-8 cells) and one l-asp-resistant cell line (KF28 cells) was evaluated by detecting LC3B form-II (LC3B-II), an autophagosome marker, via western blot analysis. LC3B-II levels were increased by l-asp treatment, and this increase was enhanced by inhibiting autophagosome turnover via the addition of a lysosome inhibitor, bafilomycin A1 (Baf), indicating that treatment with l-asp can induce autophagy equivalently in both l-asp-sensitive and insensitive cells (Figure 3E). Similarly, the induction of autophagy was also shown in both control–vector (sensitive) and GS-expressing (resistant) OVCAR-3 cells (Figure 3F). These findings exclude the possibility that the induction of autophagy is a determinant for the differential sensitivity to l-asp in OVC cells. Taken together, the depletion of Gln using l-asp may be therapeutically useful strategy for OVCs with downregulation of GS expression. Effect of GS expression on tumor growth in vitro and in vivo Finally, we found that re-expression of GS induced the inhibition of cell growth under 2D culture or three-dimensional (3D) spheroid culture with standard medium in GSlow-OVCAR-3 cells (Figure 4A). This GS-mediated growth inhibition was also shown under the hypoxic condition (Figure 4B). Furthermore, an in vivo tumor growth assay with tumors formed by the injection of OVC cells into the subcutaneous space of nude mice revealed that the re-expression of the GS protein inhibited tumor growth by OVCAR-3 and OVCAR-8 cells (Figure 4C and D). In addition, in the peritoneal dissemination model using ES-2 cells, derived from an ovarian clear cell carcinoma, dissemination was frequently observed in the peritoneal cavity with the control–vector cells, whereas none was observed with the ES-2 cells that stably expressed GS (Figure 4E). These findings suggest that the downregulation of GS expression may be associated with tumorigenesis in OVC. Figure 4. View largeDownload slide Effect of GS expression on tumor growth in vitro and in vivo. (A) Cell growth assay with 2D or 3D culture in vitro. Flag-tagged empty vector (Vec) or FLAG-tagged GS-expressing OVCAR-3 cells were cultured with the standard medium. Left panel: the relative cell growth ratio for the 2D culture at 3 days is indicated. The error bars are not visualized because they are too small. Right panel: the relative spheroid diameter size in the 3D spheroid culture at 10 days is indicated. Bar, SD. *P < 0.05. (B) Cell survival assay under hypoxic conditions. Flag-tagged empty vector (Vec) or FLAG-tagged GS-expressing OVCAR-3 cells were cultured under normoxic or hypoxic conditions in the standard medium for 24 or 48 h. Cell survival rates under the hypoxic condition relative to that under the normoxic condition are indicated. Bar, SD. *P < 0.05. (C) In vivo tumorigenesis assay using subcutaneous injection. Flag-tagged empty vector (Vec) or GS-expressing OVCAR-3 cells were subcutaneously injected into nude mice, and the tumors were resected at 14 days after injection. Image (upper) and tumor weight (lower) of resected tumors are indicated. *P < 0.05. (D) In vivo tumorigenesis assay using subcutaneous injection. Flag-tagged empty vector (Vec) or GS-expressing OVCAR-8 cells were subcutaneously injected into nude mice. Images at 14 days after injection are shown. The tumor incidence was five of six for mice with the vector cells, whereas none of six mice with the GS-expressing cells had tumors. The white arrows indicate the engrafted tumors for the vector cells. (E) In vivo tumorigenesis assay using intraperitoneal injection. Left panel: western blot analysis of the GS protein. Flag-tagged empty vector (Vec) or GS-expressing ES-2 cells were intraperitoneally injected into nude mice as a tumor dissemination model. Right panel: Images indicate dissemination in the peritoneal cavity of mice at 14 days after injection. Tumor dissemination was observed in all three of the mice with vector cells but none of the three mice with GS-expressing cells. Areas enclosed by the white dotted line indicate the engrafted tumors for the vector cells. Figure 4. View largeDownload slide Effect of GS expression on tumor growth in vitro and in vivo. (A) Cell growth assay with 2D or 3D culture in vitro. Flag-tagged empty vector (Vec) or FLAG-tagged GS-expressing OVCAR-3 cells were cultured with the standard medium. Left panel: the relative cell growth ratio for the 2D culture at 3 days is indicated. The error bars are not visualized because they are too small. Right panel: the relative spheroid diameter size in the 3D spheroid culture at 10 days is indicated. Bar, SD. *P < 0.05. (B) Cell survival assay under hypoxic conditions. Flag-tagged empty vector (Vec) or FLAG-tagged GS-expressing OVCAR-3 cells were cultured under normoxic or hypoxic conditions in the standard medium for 24 or 48 h. Cell survival rates under the hypoxic condition relative to that under the normoxic condition are indicated. Bar, SD. *P < 0.05. (C) In vivo tumorigenesis assay using subcutaneous injection. Flag-tagged empty vector (Vec) or GS-expressing OVCAR-3 cells were subcutaneously injected into nude mice, and the tumors were resected at 14 days after injection. Image (upper) and tumor weight (lower) of resected tumors are indicated. *P < 0.05. (D) In vivo tumorigenesis assay using subcutaneous injection. Flag-tagged empty vector (Vec) or GS-expressing OVCAR-8 cells were subcutaneously injected into nude mice. Images at 14 days after injection are shown. The tumor incidence was five of six for mice with the vector cells, whereas none of six mice with the GS-expressing cells had tumors. The white arrows indicate the engrafted tumors for the vector cells. (E) In vivo tumorigenesis assay using intraperitoneal injection. Left panel: western blot analysis of the GS protein. Flag-tagged empty vector (Vec) or GS-expressing ES-2 cells were intraperitoneally injected into nude mice as a tumor dissemination model. Right panel: Images indicate dissemination in the peritoneal cavity of mice at 14 days after injection. Tumor dissemination was observed in all three of the mice with vector cells but none of the three mice with GS-expressing cells. Areas enclosed by the white dotted line indicate the engrafted tumors for the vector cells. Discussion Gln is a resource for the synthesis of many AAs, proteins, nucleotides and other biologically important molecules and provides nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH) to maintain redox homeostasis (23–25). Thus, Gln plays a critical role in cell growth and cell survival. Because many cancer cell lines cannot survive without an exogenous supply of Gln, it has been believed that these cells are dependent on the uptake of extracellular Gln, even though Gln is an NEAA that can be synthesized endogenously (23). However, in this study, we showed that the depletion of extracellular Gln is effective for cell growth suppression in GSlow-OVC cells but not in GShigh-OVC cells. Others have previously reported on the GS dependency of the sensitivity to Gln depletion of cell lines from glioblastoma and breast cancer (26,27). Thus, the Gln level endogenously produced by GS is sufficient as a biological resource for GShigh-OVC cells to survive despite the depletion of extracellular Gln. It has been reported that GS is highly expressed in human cancers, including hepatocellular carcinoma, glioblastoma and breast cancer, and can support the cell survival and proliferation of those types of cancer cells (27–30). Conversely, we showed that the expression of the GS protein was downregulated in a subset of primary OVC tumors. Notably, the re-expression of GS in GSlow-OVC cells can inhibit tumor growth in vitro and in vivo. However, the detailed molecular mechanism of this phenomenon currently remains unknown. Gln can support the survival and growth of cancer cells not only by the synthesis of nucleotides and proteins but also by the production of energy from tricarboxylic acid (TCA) cycle metabolites via the conversion to Glu and α-ketoglutarate through the activity of multiple enzymes (glutaminolysis) (23). Therefore, we consider that the impaired production of endogenous Gln by low expression of GS may contribute to tumorigenesis by activating the extracellular Gln-mediated glutaminolysis for cell survival and cell growth as shown in Figure 5. The re-expression of GS into the GSlow-OVC cells may lead to metabolic restoration accompanied by reduced glutaminolysis, resulting in the reduction of activity for cell growth under the standard cell culture condition in vitro and tumor growth in vivo. Further investigation, including assessing the change in the number of metabolic intermediates with the re-expression of GS, will be required to define the biological significance of the downregulation of GS expression for the cell growth and survival of OVC cells. Figure 5. View largeDownload slide Potential of extracellular Gln-depletion therapy for GSlow-OVCs. High-GS-expressing OVC cells (GShigh-OVC cells) can survive via the GS-mediated synthesis of endogenous Gln (upper left), even under extracellular Gln-depleted conditions as induced by treatment with l-asp (middle left). By contrast, low-GS-expressing OVC cells (GSlow-OVC cells) are dependent on extracellular Gln (upper right). Thus, Gln-depletion therapy with l-asp may be potentially useful against GSlow-OVC cells (middle right). GS re-expression in GSlow-OVC cells leads to the inhibition of cell growth (lower right). Figure 5. View largeDownload slide Potential of extracellular Gln-depletion therapy for GSlow-OVCs. High-GS-expressing OVC cells (GShigh-OVC cells) can survive via the GS-mediated synthesis of endogenous Gln (upper left), even under extracellular Gln-depleted conditions as induced by treatment with l-asp (middle left). By contrast, low-GS-expressing OVC cells (GSlow-OVC cells) are dependent on extracellular Gln (upper right). Thus, Gln-depletion therapy with l-asp may be potentially useful against GSlow-OVC cells (middle right). GS re-expression in GSlow-OVC cells leads to the inhibition of cell growth (lower right). Our data revealed that inhibiting the uptake or decreasing the amount of extracellular Gln may be a therapeutically useful strategy for patients with GSlow-OVC cells (Figure 5). Treatment with l-asp is a physiologically safe strategy to induce the enzymatic lowering of extracellular Gln because this enzyme can catalyze the hydrolysis not only of asparagine to aspartate but also of Gln to Glu. We demonstrated that treatment with l-asp was more effective in the GSlow-OVC cells than in the GShigh-OVC cells. Consistently, the inhibition of GS function by treatment with methionine-l-sulfoximine, which is known as a GS inhibitor, has enhanced l-asp-induced cytotoxicity in hepatocellular carcinoma cells (31). An alternative agent that could be used to deplete plasma Gln is phenylbutyrate, an FDA-approved pharmacologic for the treatment of hyperammonemia in patients with congenital urea cycle disorders (32). Pharmacologic doses of phenylbutyrate lead to a significant depletion of plasma Gln levels by conjugating with Gln via Gln-acyltransferase to yield phenylacetylglutamine (33,34). Furthermore, in our study, the siRNA-mediated inhibition of a Gln transporter, ASCT2, was more effective in the GSlow-OVC cells than in the GShigh-OVC cells; therefore, suppressing Gln uptake may be therapeutically applicable for GSlow-OVC cells (29,35,36). Indeed, l-γ-glutamyl-p-nitroanilide (GPNA), one of a panel of ASCT2 inhibitors, has been reported to inhibit Gln uptake and inhibit the growth of cancer cells (37,38). Taken together, our findings that the depletion of extracellular Gln is effective in GSlow-OVC cells provide novel insights into the development of precision cancer therapy for OVC based on GS expression. Supplementary material Supplementary Figure 1 and 2 can be found at Carcinogenesis online. Funding This work was supported in part by Grant-in-Aid for Scientific Research (C:15K08301, Jun Inoue), Grant-in-Aid for Scientific Research on Innovative Areas ‘Conquering cancer through NEO-dimensional systems understandings’ (15H05908, Johji Inazawa) from JSPS, and the Joint Usage/Research Program of MRI, TMDU. Abbreviations AAs amino acids ALL acute lymphoblastic leukemia EAAs essential amino acids Gln glutamine Glu glutamate GS glutamine synthetase H-score histo-score l-asp l-asparaginase NEAAs nonessential amino acids OVC ovarian cancer PBS phosphate-buffered saline shRNA short hairpin RNA siRNAs small-interfering RNAs Acknowledgements The authors thank Ayako Takahashi and Rumi Mori (Tokyo Medical and Dental University, Japan) for their technical assistance. Conflict of Interest Statement: None declared. References 1. Ovarian epithelial, fallopian tube, and primary peritoneal cancer treatment (PDQ®): health professional version . In: PDQ Adult Treatment Editorial Board. PDQ Cancer Information Summaries [Internet] . Bethesda (MD) : National Cancer Institute (US) . 2. Karnezis , A.N. , et al. ( 2017 ) The disparate origins of ovarian cancers: pathogenesis and prevention strategies . Nat. Rev. Cancer , 17 , 65 – 74 . Google Scholar CrossRef Search ADS PubMed 3. Matulonis , U.A. , et al. ( 2016 ) Ovarian cancer . Nat. Rev. Dis. Primers , 2 , 16061 . Google Scholar CrossRef Search ADS PubMed 4. Fürst , P. , et al. ( 2004 ) What are the essential elements needed for the determination of amino acid requirements in humans ? J. Nutr ., 134 ( 6 Suppl ), 1558S – 1565S . Google Scholar CrossRef Search ADS PubMed 5. Reeds , P.J . ( 2000 ) Dispensable and indispensable amino acids for humans . J. Nutr ., 130 , 1835S – 1840S . Google Scholar CrossRef Search ADS PubMed 6. Young , V.R . ( 1994 ) Adult amino acid requirements: the case for a major revision in current recommendations . J Nutr ., 124 , 1517S – 1523S . Google Scholar CrossRef Search ADS PubMed 7. Geck , R.C. , et al. ( 2016 ) Nonessential amino acid metabolism in breast cancer . Adv. Biol. Regul ., 62 , 11 – 17 . Google Scholar CrossRef Search ADS PubMed 8. Jain , M. , et al. ( 2012 ) Metabolite profiling identifies a key role for glycine in rapid cancer cell proliferation . Science , 336 , 1040 – 1044 . Google Scholar CrossRef Search ADS PubMed 9. Loayza-Puch , F. , et al. ( 2016 ) Tumour-specific proline vulnerability uncovered by differential ribosome codon reading . Nature , 530 , 490 – 494 . Google Scholar CrossRef Search ADS PubMed 10. Maddocks , O.D. , et al. ( 2013 ) Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells . Nature , 493 , 542 – 546 . Google Scholar CrossRef Search ADS PubMed 11. Qiu , F. , et al. ( 2014 ) Arginine starvation impairs mitochondrial respiratory function in ASS1-deficient breast cancer cells . Sci. Signal ., 7 , ra31 . Google Scholar CrossRef Search ADS PubMed 12. Son , J. , et al. ( 2013 ) Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway . Nature , 496 , 101 – 105 . Google Scholar CrossRef Search ADS PubMed 13. Sheen , J.H. , et al. ( 2011 ) Defective regulation of autophagy upon leucine deprivation reveals a targetable liability of human melanoma cells in vitro and in vivo . Cancer Cell , 19 , 613 – 628 . Google Scholar CrossRef Search ADS PubMed 14. Oettgen , H.F. , et al. ( 1967 ) Inhibition of leukemias in man by L-asparaginase . Cancer Res ., 27 , 2619 – 2631 . Google Scholar PubMed 15. Richards , N.G. , et al. ( 2006 ) Asparagine synthetase chemotherapy . Annu. Rev. Biochem ., 75 , 629 – 654 . Google Scholar CrossRef Search ADS PubMed 16. Kikuchi , R. , et al. ( 2007 ) Promoter hypermethylation contributes to frequent inactivation of a putative conditional tumor suppressor gene connective tissue growth factor in ovarian cancer . Cancer Res ., 67 , 7095 – 7105 . Google Scholar CrossRef Search ADS PubMed 17. Nozawa , S. , et al. ( 1988 ) Establishment of a human ovarian clear cell carcinoma cell line (RMG-I) and its single cell cloning—with special reference to the stem cell of the tumor . Hum. Cell , 1 , 426 – 435 . Google Scholar PubMed 18. Iwadate , R. , et al. ( 2014 ) High expression of SQSTM1/p62 protein is associated with poor prognosis in epithelial ovarian cancer . Acta Histochem. Cytochem ., 47 , 295 – 301 . Google Scholar CrossRef Search ADS PubMed 19. Numata , M. , et al. ( 2013 ) The clinical significance of SWI/SNF complex in pancreatic cancer . Int. J. Oncol ., 42 , 403 – 410 . Google Scholar CrossRef Search ADS PubMed 20. Ananieva , E . ( 2015 ) Targeting amino acid metabolism in cancer growth and anti-tumor immune response . World J. Biol. Chem ., 6 , 281 – 289 . Google Scholar CrossRef Search ADS PubMed 21. Krall , A.S. , et al. ( 2015 ) Rethinking glutamine addiction . Nat. Cell Biol ., 17 , 1515 – 1517 . Google Scholar CrossRef Search ADS PubMed 22. Takahashi , H. , et al. ( 2017 ) Autophagy is required for cell survival under L-asparaginase-induced metabolic stress in acute lymphoblastic leukemia cells . Oncogene , 36 , 4267 – 4276 . Google Scholar CrossRef Search ADS PubMed 23. Altman , B.J. , et al. ( 2016 ) From Krebs to clinic: glutamine metabolism to cancer therapy . Nat. Rev. Cancer , 16 , 749 . Google Scholar CrossRef Search ADS PubMed 24. Hosios , A.M. , et al. ( 2016 ) Amino acids rather than glucose account for the majority of cell mass in proliferating mammalian cells . Dev. Cell , 36 , 540 – 549 . Google Scholar CrossRef Search ADS PubMed 25. Wise , D.R. , et al. ( 2008 ) Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction . Proc. Natl. Acad. Sci. USA , 105 , 18782 – 18787 . Google Scholar CrossRef Search ADS 26. Chiu , M. , et al. ( 2014 ) Glutamine depletion by crisantaspase hinders the growth of human hepatocellular carcinoma xenografts . Br. J. Cancer , 111 , 1159 – 1167 . Google Scholar CrossRef Search ADS PubMed 27. Bott , A.J. , et al. ( 2015 ) Oncogenic Myc induces expression of glutamine synthetase through promoter demethylation . Cell Metab ., 22 , 1068 – 1077 . Google Scholar CrossRef Search ADS PubMed 28. Kung , H.N. , et al. ( 2011 ) Glutamine synthetase is a genetic determinant of cell type-specific glutamine independence in breast epithelia . PLoS Genet ., 7 , e1002229 . Google Scholar CrossRef Search ADS PubMed 29. Jin , L. , et al. ( 2016 ) Glutaminolysis as a target for cancer therapy . Oncogene , 35 , 3619 – 3625 . Google Scholar CrossRef Search ADS PubMed 30. Tardito , S. , et al. ( 2015 ) Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma . Nat. Cell Biol ., 17 , 1556 – 1568 . Google Scholar CrossRef Search ADS PubMed 31. Tardito , S. , et al. ( 2011 ) L-Asparaginase and inhibitors of glutamine synthetase disclose glutamine addiction of β-catenin-mutated human hepatocellular carcinoma cells . Curr. Cancer Drug Targets , 11 , 929 – 943 . Google Scholar CrossRef Search ADS PubMed 32. Enns , G.M. , et al. ( 2007 ) Survival after treatment with phenylacetate and benzoate for urea-cycle disorders . N. Engl. J. Med ., 356 , 2282 – 2292 . Google Scholar CrossRef Search ADS PubMed 33. Thibault , A. , et al. ( 1995 ) Phase I study of phenylacetate administered twice daily to patients with cancer . Cancer , 75 , 2932 – 2938 . Google Scholar CrossRef Search ADS PubMed 34. Darmaun , D. , et al. ( 1998 ) Phenylbutyrate-induced glutamine depletion in humans: effect on leucine metabolism . Am. J. Physiol ., 274 ( 5 Pt 1 ), E801 – E807 . Google Scholar PubMed 35. van Geldermalsen , M. , et al. ( 2016 ) ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer . Oncogene , 35 , 3201 – 3208 . Google Scholar CrossRef Search ADS PubMed 36. Wise , D.R. , et al. ( 2010 ) Glutamine addiction: a new therapeutic target in cancer . Trends Biochem. Sci ., 35 , 427 – 433 . Google Scholar CrossRef Search ADS PubMed 37. Fuchs , B.C. , et al. ( 2005 ) Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime ? Semin. Cancer Biol ., 15 , 254 – 266 . Google Scholar CrossRef Search ADS PubMed 38. Esslinger , C.S. , et al. ( 2005 ) Ngamma-aryl glutamine analogues as probes of the ASCT2 neutral amino acid transporter binding site . Bioorg. Med. Chem ., 13 , 1111 – 1118 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
TI - Ovarian cancer therapeutic potential of glutamine depletion based on GS expression
JF - Carcinogenesis
DO - 10.1093/carcin/bgy033
DA - 2018-04-02
UR - https://www.deepdyve.com/lp/oxford-university-press/ovarian-cancer-therapeutic-potential-of-glutamine-depletion-based-on-eCn0DAZSeH
SP - 1
EP - 766
VL - Advance Article
IS - 6
DP - DeepDyve
ER -