Endogenous glutamate determines ferroptosis sensitivity via ADCY10-dependent YAP suppression in lung adenocarcinoma

Rationale: Ferroptosis, a newly identified form of regulated cell death, can be induced following the inhibition of cystine-glutamate antiporter system XC- because of the impaired uptake of cystine. However, the outcome following the accumulation of endogenous glutamate in lung adenocarcinoma (LUAD) has not yet been determined. Yes-associated protein (YAP) is sustained by the hexosamine biosynthesis pathway (HBP)-dependent O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation), and glutamine-fructose-6-phosphate transaminase (GFPT1), the rate-limiting enzyme of the HBP, can be phosphorylated and inhibited by adenylyl cyclase (ADCY)-mediated activation of protein kinase A (PKA). However, whether accumulated endogenous glutamate determines ferroptosis sensitivity by influencing the ADCY/PKA/HBP/YAP axis in LUAD cells is not understood. Methods: Cell viability, cell death and the generation of lipid reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to evaluate the responses to the induction of ferroptosis following the inhibition of system XC-. Tandem mass tags (TMTs) were employed to explore potential factors critical for the ferroptosis sensitivity of LUAD cells. Immunoblotting (IB) and quantitative RT-PCR (qPCR) were used to analyze protein and mRNA expression. Co-immunoprecipitation (co-IP) assays were performed to identify protein-protein interactions and posttranslational modifications. Metabolite levels were measured using the appropriate kits. Transcriptional regulation was evaluated using a luciferase reporter assay, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA). Drug administration and limiting dilution cell transplantation were performed with cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. The associations among clinical outcome, drug efficacy and ADCY10 expression were determined based on data from patients who underwent curative surgery and evaluated with patient-derived primary LUAD cells and tissues. Results: The accumulation of endogenous glutamate following system XC- inhibition has been shown to determine ferroptosis sensitivity by suppressing YAP in LUAD cells. YAP O-GlcNAcylation and expression cannot be sustained in LUAD cells upon impairment of GFPT1. Thus, Hippo pathway-like phosphorylation and ubiquitination of YAP are enhanced. ADCY10 acts as a key downstream target and diversifies the effects of glutamate on the PKA-dependent suppression of GFPT1. We also discovered that the protumorigenic and proferroptotic effects of ADCY10 are mediated separately. Advanced-stage LUADs with high ADCY10 expression are sensitive to ferroptosis. Moreover, LUAD cells with acquired therapy resistance are also prone to higher ADCY10 expression and are more likely to respond to ferroptosis. Finally, a varying degree of secondary labile iron increase is caused by the failure to sustain YAP-stimulated transcriptional compensation for ferritin at later stages further explains why ferroptosis sensitivity varies among LUAD cells. Conclusions: Endogenous glutamate is critical for ferroptosis sensitivity following the inhibition of system XC- in LUAD cells, and ferroptosis-based treatment is a good choice for LUAD patients with later-stage and/or therapy-resistant tumors.

(D) Schematic representation shows how β-ME and NAC promotes GSH synthesis. p-YAP S127 , O-YAP T241 and total YAP were measured by IB in H1975 cells treated with or without erastin (10 μM), in the presence or absence of β-ME (50 μM), NAC (1 mM) or GSH (50 μM) for 8 h. The level of proteins was normalized to that of GAPDH, and the normalized level of proteins in DMSO-treated cells was arbitrarily set to 1.
(E) Overexpressing transaminases has no roles on glutamate. GOT2 and overexpression (left). Intracellular glutamate was also measured and graphed (right).
(F) Endogenous glutamate is suppressed by overexpressing GLUD1 with simultaneous knocking GLAST and GLT1 down. GLUD1, GLAST and GLT1 were measured by IB in H1975 cells with indicated treatment (left). Intracellular glutamate was also measured and graphed (right).
(G) GLUD1, GLAST and GLT1 were measured by IB in H1975-based GGG cells with or without Dox (1 µg/ml) treatment for 24 h.
(H-K) Dox has no roles on cell viability, cell death and lipid peroxidation, as evaluating by cellular ATP (H), SYTOX green (I) and C11BODIPY (J) probing followed by flow cytometry, and MDA concentration (K) in LUAD cells after treating with or without Dox (1 µg/ml) for 24 h in LUAD cells.
(L) Adding excessive exogenous glutamate is ineffective to restore endogenous glutamate in Dox-treated H1975-based GGG cells. H1975-based GGG cells were pretreated with or without Dox (1 µg/ml) for 24 h before further treating with exogenous glutamate at indicated concentration for 24 h. Intracellular glutamate was measured and graphed.
(P) Endogenous glutamate has no effect on cell proliferation during the timeframe of our treatment. Relative cell number was monitored at indicated hours in H1975-based GGG cells treated with or without Dox (1 µg/ml) and EGCG (5 μM). The initial cell number was 10 5 per well.
(Q) Intracellular glutamine was measured in indicated LUAD cells with or without erastin treatment (10 μM) for 24 h.
(R) α-KG has no roles on YAP modification and expression. p-YAP S127 , O-YAP T241 and total YAP were measured by IB in H1975 cells treated with or without erastin (10 μM), in the presence or absence of AOA (1 mM) and DMK (4 mM) for 8 h. The level of proteins was normalized to that of GAPDH, and the normalized level of proteins in DMSO-treated cells was arbitrarily set to 1.
The data are shown as the mean ±SD from three biological replicates (including IB). **P < 0.01 indicates statistical significance. Data in A, E, F, L, O, Q were analyzed using a one-way ANOVA test. Data in B, C, H, I, J, K, M, P were analyzed using a two-way ANOVA test. (B) GFPT1 S205A compensates reduced GFPT1 activity by erastin.

Supplementary
FLAG-GFPT1 S205A was ectopically expressed in H1975 cells prior to the treatment with or without erastin (10 μM) for 8 h. GFPT1 activity was measured using GDH method.
(D) FLAG-GFPT1 S205A was ectopically expressed in H1975 cells prior to the treatment with or without Sorafenib (5 μM) for 8 h. GFPT1 activity was measured using GDH method.
The data are shown as the mean ±SD from three biological replicates. *P < 0.05, **P < 0.01 indicates statistical significance. All the data in were analyzed using a one-way ANOVA test. (N) ADCY10 mRNA was measured in indicated LUAD cells by qPCR, and its correlation with reduced GFPT1 activity (calculated as percentage of erastin treating group to the ones treated with DMSO) was analyzed using the Spearman rank-correlation analysis.
(O) Intracellular glutamate was measured in the same specimens as those in Figure 5N after treating with DMSO or erastin (10 μM) for 24 h.
The data are shown as the mean ±SD from three biological replicates (including IB). **P < 0.01 indicates statistical significance. Data in E, G, L, M, O were analyzed using a one-way ANOVA test. Data in D, J were analyzed using a Student's t test. Data in I were analyzed using a two-way ANOVA test. Data in N were analyzed using the Spearman rank-correlation analysis. (B) Colony formation was analyzed by anchorage-independent soft agar assay in H1975 cells with or without ADCY10 knockdown. Relative colony size and amounts (Φ >30 μm) were counted and graphed on the right. Scale bar, 50 μm.

Supplementary
(C) 3D spheroids generated from H1975 cells with or without ADCY10 knockdown. Relative spheroid size and amounts (Φ >30 μm) were counted and graphed on the right. Scale bar, 50 μm.
(D) Representative images of xenografts that formed at the endpoint of the experiments which were the same as those in Figure 6A. Scale bar, 5 mm.
(E) MDA was measured in xenografts which were formed in Figure 6A  The data are shown as the mean ±SD from three biological replicates (including IB). **P < 0.01 indicates statistical significance. Data in B, C, E, G, H were analyzed using a one-way ANOVA test. Data in I were analyzed using a two-way ANOVA test. (K) A TFCP2 motif (image from JASPAR database) was identified within human FTH1 promoter (-157~-148 bp relative to TSS) as predicted using FIMO software.
(L) Nuclear extracts from H1975 cells were incubated with anti-IgG or anti-TFCP2 antibodies for 15min prior to further incubating with biotin-labeled probes containing TFCP2 motif from FTH1 promoter. The TFCP2-DNA complexes and supershifts were measured using EMSA.
(M) Enrichments of TFCP2 but not CREB at TFCP2 motif within the FTH1 promoter were measured in H1975 cells using ChIP followed by agarose gel electrophoresis. Regions at -2k/+2k were used as negative controls.
(N) The luciferase activities of FTH1 promoter with or without TFCP2 motif mutation were measured in H1975 cells ectopically expressed with TFCP2 and YAP.
(O) Co-occupancy of YAP and TFCP2 within the FTH1 promoter were measured in H1975 cells with or without erastin (10 μM) or Sorafenib (5 μM) treatment for 4 h using Re-ChIP followed by agarose gel electrophoresis.
(Q) NCOA4 knockdown efficiency in H1975 cells transfected with or without shRNAs targeting against NCOA4.