Argininosuccinate Synthetase 1 Loss in Invasive Bladder Cancer Regulates Survival through General Control Nonderepressible 2 Kinase-Mediated Eukaryotic Initiation Factor 2α Activity and Is Targetable by Pegylated Arginine Deiminase

Abstract

Loss of argininosuccinate synthetase 1 (ASS1), a key enzyme for arginine synthesis, occurs in many cancers, making cells dependent on extracellular arginine and targetable by the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20). We evaluated ASS1 expression and effects of ASS1 loss in bladder cancer which, despite affecting >70,000 people in the United States annually, has limited therapies. ASS1 loss was identified in conventional and micropapillary urothelial carcinoma, small cell, and squamous cell carcinoma subtypes of invasive bladder cancer, as well as in T24, J82, and UM-UC-3 but not in 5637, RT112, and RT4 cell lines. ASS1-deficient cells showed preferential sensitivity to ADI-PEG 20, evidenced by decreased colony formation, reduced cell viability, and increased sub-G1 fractions. ADI-PEG 20 induced general control nonderepressible 2-dependent eukaryotic initiation factor 2α phosphorylation and activating transcription factor 4 and C/EBP homologous protein up-regulation, associated with caspase-independent apoptosis and autophagy. These effects were ablated with selective siRNA silencing of these proteins. ASS1 overexpression in UM-UC-3 or ASS1 silencing in RT112 cells reversed these effects. ADI-PEG 20 treatment of mice bearing contralateral flank UM-UC-3 and RT112 xenografts selectively arrested tumor growth in UM-UC-3 xenografts, which had reduced tumor size, reduced Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. This suggests that ASS1 loss occurs in invasive bladder cancer and is targetable by ADI-PEG 20.

Figure 1

ASS1 expression is reduced in some human bladder cancers and cell lines. A: Normal urothelium, urothelial carcinoma, and squamous cell carcinoma were assessed for ASS1 expression by immunohistochemistry. B: The percentage of cells showing 0 or 1+ ASS1 expression (on a scale of 0–3) is shown for a series of Nl and invasive carcinomas, consisting of Adeno, MP, UCa, SCC, and Squam. C: Box plots representing relative mRNA expression levels of ASS1 and ASL in human bladder tumors compared with normal, from the TCGA database. The data for ASL were split into two subcolumns because two probes map onto the same gene. D: Overall patient survival plotted related to ASS1 expression; high ASS1 (above median) versus low ASS1 (below median). E: Representative immunoblot of ASS1 in benign and malignant bladder cells; Jurkat cells were used as a positive control. n = 19 Nl (B); n = 19 Adeno (B); n = 27 MP (B); n = 148 UCa (B); n = 19 SCC (B); n = 39 Squam (B). ^∗P < 0.05 compared with normal urothelium, Fishers test (B); ^∗P < 0.05 compared with normal, Wilcoxon test (C); P = 0.0082, log-rank (Mantel Cox) text, P = 0.0072, Gehan-Breslow-Wilcoxon text (D). Original magnification, ×100 (A). Adeno, adenocarcinoma; ASL, argininosuccinate lyase; ASS1, argininosuccinate synthetase 1; Ca, carcinoma; H&E, hematoxylin and eosin; IHC, immunohistochemistry; MP, micropapillary urothelial carcinoma; Nl, normal urothelium; SCC, small cell carcinoma; Squam, squamous cell carcinoma; TCGA, the Cancer Genome Atlas Project; UCa, urothelial carcinoma.

Figure 2

ADI-PEG 20 reduces colony formation growth and cell viability in ASS1-deficient bladder cancer cells. A: Representative images are shown for colony formation in J82 and RT112 cells treated with increasing concentrations of ADI-PEG 20 for 6 to 10 days. B: Quantification of surviving fraction of colonies from colony formation assays in UM-UC-3, J82, RT112, and 5637 cells. C: Results from three independent MTT assays to calculate IC₅₀ and AUC. D–G: Cell viability in UM-UC-3, J82, RT112, and 5637 cells, respectively, treated with increasing concentrations of ADI-PEG 20 for 48 hours. ^∗P < 0.05 compared with untreated control, t-test. ADI-PEG 20, pegylated arginine deiminase; ASS1, argininosuccinate synthetase 1; AUC, area under the curve; IC₅₀, concentration that inhibits 50%.

Figure 3

Arginine deprivation therapy leads to caspase-independent apoptotic cell death in ASS1-deficient cells. A: PI-based cell-cycle analysis in UM-UC-3 and RT112 cells treated with increasing concentrations of ADI-PEG 20 for 48 hours. B: PI/annexin V flow cytometric analysis in UM-UC-3 and RT112 cells treated with increasing concentrations of ADI-PEG 20 for 48 hours. C: Quantification of PI/annexin V flow cytometric analysis of UM-UC-3, J82, RT112, and 5637 bladder cancer cells. D and E: Immunoblot of PARP and caspase cleavage in ASS1-deficient and -expressing cell lines treated with increasing concentrations of ADI-PEG 20 for 48 hours. Cytochrome C lysate was used as positive control. Data from a single experiment, representative of three independent experiments are presented (B). ADI-PEG 20, pegylated arginine deiminase; ASS1, argininosuccinate synthetase 1; Cyt C, cytochrome C; PARP, poly (ADP-ribose) polymerase; NA, not applicable; PI, propidium iodide.

Figure 4

Arginine deprivation or ADI-PEG 20 treatment leads to autophagy in ASS1-deficient bladder cancer cells. A: Representative immunoblots of LC3I and LC3II in UM-UC-3, J82, RT112, and 5637 cells after ADI-PEG 20 administration alone, or with CQ, for 6 hours. Arginine-free media were used as arginine deprivation control. B: LC3 punctae were increased in UM-UC-3 and J82 cells on ADI-PEG 20 or CQ treatment for 6 hours, compared with untreated cells. Co-incubation of cells with both ADI-PEG 20 and CQ caused the greatest increase in punctae. DAPI was used to counterstain the nucleus. ADI-PEG 20, pegylated arginine deiminase; ASS1, argininosuccinate synthetase 1; CQ, chloroquine.

Figure 5

Arginine deprivation or ADI-PEG 20 treatment leads to eIF2α phosphorylation (Ser51), ATF4-CHOP induction, and LC3II accumulation in ASS1-deficient bladder cancer cells. A: Immunoblot of p-eIF2α (Ser51), ATF4, and CHOP in UM-UC-3, J82, RT112 and 5637 cells after ADI-PEG 20 administration for 6 hours. Arginine-free media were used as arginine deprivation control. B and C: p-eIF2α (Ser51), ATF4, and CHOP induction, and LC3II accumulation on treatment with ADI-PEG 20 or arginine-free media for 6 hours is ablated in ASS1-overexpressing UM-UC-3 cells compared with empty vector control cells (B) and increased in ASS1-silenced RT112 cells compared with NTC control cells (C). D: Immunoblot of ASS1 shows re-expression in J82 cells on 5-Aza treatment. E: MTT assays in the aforementioned cells for 48 hours. ^∗P < 0.05 compared with respective controls (UM-UC-3 pCMV, RT112 si-NTC, or J82 DMSO) at each ADI-PEG 20 concentration, t-test. ADI-PEG 20, pegylated arginine deiminase; ASS1, argininosuccinate synthetase 1; ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; DMSO, dimethyl sulfoxide; eIF2α, eukaryotic initiation factor 2α; pCMV, cytomegalovirus plasmid; p-eIF2α, phosphorylated eukaryotic initiation factor 2α; si-NTC, nontargeting control siRNA; 5-Aza, 5-azacytidine.

Figure 6

Arginine deprivation or ADI-PEG 20 treatment leads to GCN2 kinase-mediated eIF2α phosphorylation (Ser51) in ASS1-deficient bladder cancer cells. A: J82 and UM-UC-3 cells were transfected with NTC-siRNA or GCN2-siRNA and effects on eIF2α (Ser51) phosphorylation, ATF4, CHOP, and LC3 was assessed after ADI-PEG20 or arginine-free media administration for 6 hours. B: UM-UC-3 cells were transfected with control (NTC) siRNA, ATF4-siRNA, or CHOP-siRNA, and effects on eIF2α (Ser51) phosphorylation, ATF4, CHOP, and LC3 were assessed after ADI-PEG20 or arginine-free media administration for 6 hours. C and D: LC3II/LC3I ratios in the above figures plotted by densitometry. E: Schematic of putative drug mechanism in ASS1-deficient bladder cancer cells treated with ADI-PEG 20. ADI-PEG 20, pegylated arginine deiminase; ASS1, argininosuccinate synthetase 1; ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; eIF2α, eukaryotic initiation factor 2α; GCN2, general control nonderepressible 2; NTC, nontargeting control.

Figure 7

ASS1 deficiency results in reduced tumor growth in a xenograft model of bladder cancer. A: Athymic nude mice containing UM-UC-3 xenografts (left flank) and RT112 xenografts (right flank) showed reduced UM-UC-3 volume after ADI-PEG 20 administration. B: Image of mouse xenografts after treatment with saline or ADI-PEG20. C: Grossly dissected mouse tumors after saline and ADI-PEG 20 treatment. D: H&E staining showed reduced cell numbers in ADI-PEG 20–treated tumors that correlated with reduced Ki-67 proliferation index and increased TUNEL stain. E: Quantification of Ki-67 staining. F: Quantification of TUNEL staining. ^∗P < 0.05, t-test. Original magnification, ×20 (D). ADI-PEG 20, pegylated arginine deiminase; ASS1, argininosuccinate synthetase 1; H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.