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

Abstract

Rationale: Ferroptosis, a newly identified form of regulated cell death, can be induced following the inhibition of cystine-glutamate antiporter system X C - 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 X C - . 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 X C - 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 ...

Transcriptional Repression of Ferritin Light Chain Increases Ferroptosis Sensitivity in Lung Adenocarcinoma

“…Therefore, we believe that in future clinical treatment, using ferroptotic therapy to treat drug-resistant patients or using ferroptotic therapy directly in combination with chemotherapy or targeted drugs can effectively avoid tumor progression caused by drug resistance. In addition, several ferroptotic sensitivity biomarkers (such as ACSL4, calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and ADCY10 (Doll et al, 2017;Wang et al, 2021;Zhang et al, 2021)) and therapeutic targets (such as FTL and YAP (Wu et al, 2019;Zhang et al, 2021)) have been identified, all of which provide support for ferroptosis use in clinical oncology therapy. Of course, although ferroptotic-promoting drugs such as sorafenib have been applied in clinical tumor treatment (Chen et al, 2021), the extensive application of ferroptotic therapy in clinical studies requires the guidance of clinical studies with large sample sizes and multicenter and finely grouped specimens.…”

“…In our previous study, we reported that FTH transcription first increased and then decreased after erastin treatment (Zhang et al, 2021). So how did FTL change after erastin was added?…”

“…Yes-Associated Protein/Transcription Factor CP2 Suppression Elevates Labile Iron via Ferritin Light Chain Inhibition YAP and TFCP2 are two factors affecting ferroptosis (Wu et al, 2019;Zhang et al, 2021). We further characterized the mechanism by which YAP and TFCP2 suppression promotes ferroptosis.…”

“…Abrogates Sustained Yes-Associated Protein Induced Ferroptosis Inhibition βTRCP-knockout-induced sustained YAP leads to ferroptosis inhibition (Zhang et al, 2021). Subsequently, we investigated whether sustained YAP induces ferroptosis inhibition via FTL.…”

“…A common mechanism for ferroptosis agonist-induced iron release is nuclear receptor coactivator 4 (NCOA4)-mediated autophagic degradation of ferritin, referred to as ferritinophagy (Mancias et al, 2014). Our research group also reported a mechanism that affected ferroptosis sensitivity caused by an increase in labile iron due to a disruption of the transcription balance of FTH (Zhang et al, 2021). However, whether other mechanisms by which labile iron influences ferroptosis sensitivity remains to be explored.…”