The pro-fibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression

Increased senescence and expression of pro-fibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and pro-fibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and pro-fibrotic genes were measured in primary fibroblasts from the lungs of old (24 months) and young (3 months) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53 and b-galactosidase) and increased expression of pro-fibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1 and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and pro-fibrotic genes. Young cells were induced to express the senescence and pro-fibrotic phenotype by sulfasalazine, an Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, an Slc7a11 inducer, decreased senescence without affecting pro-fibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased pro-fibrotic markers. In short, old lung fibroblasts manifest a pro-fibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

Molecular basis for redox control by the human cystine/glutamate antiporter system xc−

Cysteine plays an essential role in cellular redox homoeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc− is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc− is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc− in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.

Molecular basis for redox control by the human cystine/glutamate antiporter System xc-.

Cysteine plays an essential role in cellular redox homeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc- is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc- is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition and transport remains elusive, hampering efforts to specifically target this system. Here we present the cryo-EM structure of system xc- in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine recognition and transport in human cells.

FC 014INFLUENCE OF GENETIC VARIATION IN SLC7A13/AGT1 IN HUMAN CYSTINURIA

Background and Aims Cystinuria (CU) is an inherited renal disorder based on urinary wasting of dibasic amino acids, urinary precipitation, and consecutive cystine stone formation. It is caused by pathogenic variants in two distinct disease genes, SLC3A1 and SLC7A9, both of which encode subunits of a heterodimeric tubular amino acid transporter, rBAT/SLC3A1 and BAT1/SLC7A9, located at the apical membrane of proximal renal tubules. CU is marked by incomplete penetrance and substantial disease variability. Recently, a novel cystine transporter, consisting of the light chain AGT1/SLC7A13 and its heterodimeric partner rBAT/SLC3A1 has been identified in the S3 segment of murine proximal tubules. In this study, we aim at evaluating the role of AGT1 in cystinuric patients with or without mutations in either SLC3A1 or SLC7A9, analyzing the role of AGT1/SLC7A13 as novel disease gene or genetic modifier in CU. Method A multicenter European CU-cohort comprising 132 individuals was screened for pathogenic variants in SLC3A1, SLC7A9, and SLC7A13 using high-throughput multiplex PCR-based amplification and next-generation sequencing (MiSeq Illumina) followed by multiplex ligation-dependent probe amplification (MLPA) of SLC3A1 and SLC7A9. For functional in vitro studies, epitope-tagged human and murine rBAT and AGT1 proteins were transiently expressed in different cell systems. Heterodimer complex formation was analyzed by co-immunoprecipitation and western blot studies and membrane trafficking was evaluated by immunofluorescence microscopy. Results Genectic analysis of our CU-cohort did not reveal indiviuals with SLC7A13 variation only, however we found three patients harbouring heterozygous missense variants in addition to pathogenic or VUS variants in SLC3A1 or SLC7A9. To evaluate their influence on the generation of functional cystine transporters in vitro, different cell models were transiently transfected with plasmids expressing wildtype or mutant proteins. In line with previous reports, co-expression of AGT1 and rBAT wildtype allowed efficient complex formation as AGT1-induced maturation of rBAT was detected by increased mature N-glycosylation, co-immunoprecipitation and membrane insertion. Whereas AGT1 patient variants p.Met452Thr (SLC7A13 c.1355T>C) and p.Ile174Phe (SLC7A13 c.520A>T) behaved comparable to wildtype AGT1, variants p.Asn45Lys (SLC7A13 c.135C>G) and p.Leu270Phe (SLC7A13 c.808C>T) led to clearly reduced glycosylation patterns and trafficking deficits of rBAT wildtype protein. Next, the mutual influence of pathogenic variation in both, AGT1 and rBAT, will unravel the consequences of patient-specific molecular interactions on the functional expression of cystine transporter complexes. Conclusion Here, we report three CU-patients with variants in SLC7A13 combined with either SLC3A1 or SLC7A9. For two of these variants, in vitro functional analysis revealed pathogenic molecular mechanisms disturbing complex formation, maturation and trafficking of rBAT. We hypothesize that specific pathogenic variants in SLC7A13 interfere with efficient membrane localization of heterodimeric cystine transporters, which results in modulation of cystine transport in the S3 segment of proximal tubules in CU-patients.

PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis

Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide-sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the first time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to fight CSC chemoresistance.

Autophagy is required for proper cysteine homeostasis in pancreatic cancer through regulation of SLC7A11

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is highly refractory to current therapies. We had previously shown that PDAC can utilize its high levels of basal autophagy to support its metabolism and maintain tumor growth. Consistent with the importance of autophagy in PDAC, autophagy inhibition significantly enhances response of PDAC patients to chemotherapy in two randomized clinical trials. However, the specific metabolite(s) that autophagy provides to support PDAC growth is not yet known. In this study, we demonstrate that under nutrient-replete conditions, loss of autophagy in PDAC leads to a relatively restricted impairment of amino acid pools, with cysteine levels showing a significant drop. Additionally, we made the striking discovery that autophagy is critical for the proper membrane localization of the cystine transporter SLC7A11. Mechanistically, autophagy impairment results in the loss of SLC7A11 on the plasma membrane and increases its localization at the lysosome in an mTORC2-dependent manner. Our results demonstrate a critical link between autophagy and cysteine metabolism and provide mechanistic insights into how targeting autophagy can cause metabolic dysregulation in PDAC.

Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy

The cystine/glutamate antiporter SLC7A11 (also commonly known as xCT) functions to import cystine for glutathione biosynthesis and antioxidant defense and is overexpressed in multiple human cancers. Recent studies revealed that SLC7A11 overexpression promotes tumor growth partly through suppressing ferroptosis, a form of regulated cell death induced by excessive lipid peroxidation. However, cancer cells with high expression of SLC7A11 (SLC7A11high) also have to endure the significant cost associated with SLC7A11-mediated metabolic reprogramming, leading to glucose- and glutamine-dependency in SLC7A11high cancer cells, which presents potential metabolic vulnerabilities for therapeutic targeting in SLC7A11high cancer. In this review, we summarize diverse regulatory mechanisms of SLC7A11 in cancer, discuss ferroptosis-dependent and -independent functions of SLC7A11 in promoting tumor development, explore the mechanistic basis of SLC7A11-induced nutrient dependency in cancer cells, and conceptualize therapeutic strategies to target SLC7A11 in cancer treatment. This review will provide the foundation for further understanding SLC7A11 in ferroptosis, nutrient dependency, and tumor biology and for developing novel effective cancer therapies.

Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

ABSTRACTCancer-Associated Fibroblasts (CAFs) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression, through pro-tumour cross-talk and the generation of fibrosis (physical barrier to drugs). CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumour stroma and its prognostic significance. Herein we show that high expression of SLC7A11 in PDAC tumour stroma (but not tumour cells) is independently prognostic of poorer overall survival. We demonstrate using orthogonal approaches that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis, and that SLC7A11 inhibition significantly decreases their proliferation, reduces their resistance to oxidative stress and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, our paradigm-shifting work demonstrates the need to inhibit SLC7A11 in the PDAC stroma, as genetic ablation of SLC7A11 in PDAC cells alone is not enough to reduce tumour growth. Finally, our work validates that a nano-based gene-silencing drug against SLC7A11, developed by our group, reduces PDAC tumour growth, CAF activation and fibrosis in a mouse model of PDAC.

DYNC1LI2 regulates localization of the chaperone-mediated autophagy-receptor LAMP2A and improves cellular homeostasis in cystinosis

SUMMARYThe dynein motor protein complex is required for retrograde transport but the functions of the intermediate-light chains that form the cargo-binding complex are not elucidated and the importance of individual subunits in the maintenance of cellular homeostasis is unknown. Here, using mRNA arrays and protein analysis, we show that the dynein subunit, intermediate chain 2 (DYNC1LI2) is downregulated in cystinosis, a lysosomal storage disorder caused by genetic defects in the lysosomal cystine transporter, cystinosin. Reconstitution of the expression of DYNC1LI2 in Ctns-/- cells re-established endolysosomal dynamics. Defective vesicular trafficking in cystinotic cells was rescued by DYNC1LI2 expression which correlated with decreased endoplasmic reticulum stress manifested as decreased expression levels of the chaperone Grp78. Mitochondrial fragmentation in cystinotic fibroblasts was also rescued by DYNC1LI2. Survival of cystinotic cells to oxidative stress insult was increased by DYNC1LI2 reconstitution but not by its paralog DYNC1LI1, which also failed to decrease ER stress levels and mitochondrial fragmentation. Restoring DYNC1LI2 expression rescued the localization of the chaperone-mediated autophagy receptor, LAMP2A, and restored cellular homeostasis of cystinotic proximal tubule cells, the primary cell type affected in cystinosis. DYNC1LI2 failed to rescue phenotypes in cystinotic cells when LAMP2A was downregulated or when co-expressed with dominant negative (DN) RAB7 or DN-RAB11, which impair LAMP2A trafficking. DYNC1LI2 emerges as a new target to repair underlying trafficking and CMA defects in cystinosis, a mechanism that is not restored by currently used lysosomal depletion therapies.

Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis

BackgroundMutations in the gene that encodes the lysosomal cystine transporter cystinosin cause the lysosomal storage disease cystinosis. Defective cystine transport leads to intralysosomal accumulation and crystallization of cystine. The most severe phenotype, nephropathic cystinosis, manifests during the first months of life, as renal Fanconi syndrome. The cystine-depleting agent cysteamine significantly delays symptoms, but it cannot prevent progression to ESKD and does not treat Fanconi syndrome. This suggests the involvement of pathways in nephropathic cystinosis that are unrelated to lysosomal cystine accumulation. Recent data indicate that one such potential pathway, lysosome-mediated degradation of autophagy cargoes, is compromised in cystinosis.MethodsTo identify drugs that reduce levels of the autophagy-related protein p62/SQSTM1 in cystinotic proximal tubular epithelial cells, we performed a high-throughput screening on the basis of an in-cell ELISA assay. We then tested a promising candidate in cells derived from patients with, and mouse models of, cystinosis, and in preclinical studies in cystinotic zebrafish.ResultsOf 46 compounds identified as reducing p62/SQSTM1 levels in cystinotic cells, we selected luteolin on the basis of its efficacy, safety profile, and similarity to genistein, which we previously showed to ameliorate other lysosomal abnormalities of cystinotic cells. Our data show that luteolin improves the autophagy–lysosome degradative pathway, is a powerful antioxidant, and has antiapoptotic properties. Moreover, luteolin stimulates endocytosis and improves the expression of the endocytic receptor megalin.ConclusionsOur data show that luteolin improves defective pathways of cystinosis and has a good safety profile, and thus has potential as a treatment for nephropathic cystinosis and other renal lysosomal storage diseases.