Cystine Uptake Research Articles

315 PPAR-γ promotes cystine uptake to prevent unchecked lipid ROS and cell death in Th9 cells

Pathogenic Th2 cells are key mediators of type-2-driven skin disease. Current treatments inhibit the activation of pathogenic Th2 cells or neutralize their effector molecules. Yet, therapies targeting the long-term survival of pathogenic Th2 cells in the skin are missing. Th9 cells are a subset of IL-9-expressing effector Th cells that share overlapping features with pathogenic Th2 cells. Therefore, a better characterization of the Th9 subset is of utmost importance to identify regulators of cell survival that are specifically expressed by these cells and that might represent novel therapeutic targets.

Identification of the ultrahigh-risk subgroup in neuroblastoma cases through DNA methylation analysis and its treatment exploiting cancer metabolism

Neuroblastomas require novel therapies that are based on the exploitation of their biological mechanism. To address this need, we analyzed the DNA methylation and expression datasets of neuroblastomas, extracted a candidate gene characterizing the aggressive features, and conducted functional studies. Based on the DNA methylation data, we identified a subgroup of neuroblastoma cases with 11q loss of heterozygosity with extremely poor prognosis. PHGDH, a serine metabolism-related gene, was extracted as a candidate with strong expression and characteristic methylation in this subgroup as well as in cases with MYCN amplification. PHGDH inhibition suppressed neuroblastoma cell proliferation in vitro and in vivo, indicating that the inhibition of serine metabolism by PHGDH inhibitors is a therapeutic alternative for neuroblastoma. Inhibiting the arginine metabolism, which is closely related to serine metabolism using arginine deiminase, had a combination effect both in vitro and in vivo, especially on extracellular arginine-dependent neuroblastoma cells with ASS1 deficiency. Expression and metabolome analyses of post-dose cells confirmed the synergistic effects of treatments targeting serine and arginine indicated that xCT inhibitors that inhibit cystine uptake could be candidates for further combinatorial treatment. Our results highlight the rational therapeutic strategy of targeting serine/arginine metabolism for intractable neuroblastoma.

Cystine Induced-mTORC2 Activation through Promoting Sin1 Phosphorylation to Suppress Cancer Cell Ferroptosis.

Mechanistic target of rapamycin (mTOR) serves as a central signaling node in the coordination of cell growth and metabolism, and it functions via two distinct complexes, namely, mTOR complex 1 (mTORC1) and mTORC2. mTORC1 plays a crucial role in sensing amino acids, whereas mTORC2 involves in sensing growth factors. However, it remains largely unclear whether mTORC2 can sense amino acids and the mechanism by which amino acids regulate mTORC2 has not been studied. After treating cells with indicated concentration of amino acidsfor different time, it is found that the mTORC2 activation is significantly increased in response to amino acids stimulation, especially cystine. Particularly, knockdown solute carrier family 7 member 11 (SLC7A11)by siRNA shows that SLC7A11-mediated cystine uptake is responsible for activating mTORC2. Mechanistically, the study finds that p38 is activated in response to cystine stimulation, and co-immunoprecipitation (Co-IP) experiments suggest that p38 regulates the assembly of components within mTORC2 by mediating the phosphorylation of the mTORC2 subunit mitogen-activated protein kinase-interacting protein 1 (Sin1) in a cystine-dependent manner. Finally, combined with inducers and inhibitors of ferroptosis and cell viability assay, the study observes that cystine-mediated regulation of the p38-Sin1-mTOR-AKT pathway induces resistance to ferroptosis. These results indicate that cystine-induced activation of the p38-Sin1-mTORC2-AKT pathway suppresses ferroptosis.

SHP-1/STAT3-Signaling-Axis-Regulated Coupling between BECN1 and SLC7A11 Contributes to Sorafenib-Induced Ferroptosis in Hepatocellular Carcinoma.

Ferroptosis is a type of iron-dependent cell death pertaining to an excess of lipid peroxidation. It has been suggested that sorafenib—an anti-angiogenic medication for hepatocellular carcinoma (HCC)—induces ferroptosis, but the underlying mechanism for this remains largely unknown. We employed siRNA-mediated gene silencing to investigate the role of Src homology region 2 domain-containing phosphatase-1 (SHP-1), following sorafenib treatment, in cystine/glutamate-antiporter-system-Xc−-regulated cystine uptake. Co-immunoprecipitation was also performed to examine the interactions between MCL1, beclin 1 (BECN1), and solute carrier family 7 member 11 (SLC7A11), which functions as the catalytic subunit of system Xc−. The results of this study showed that sorafenib enhanced the activity of SHP-1, dephosphorylated STAT3, downregulated the expression of MCL1 and, consequently, reduced the association between MCL1 and BECN1. In contrast, increased binding between BECN1 and SLC7A11 was observed following sorafenib treatment. The elevated interaction between BECN1 and SLC7A11 inhibited the activity of system Xc−, whereas BECN1 silencing restored cystine intake and protected cells from ferroptosis. Notably, ectopic expression of MCL1 uncoupled BECN1 from SLC7A11 and rescued cell viability by attenuating lipid peroxidation. The results revealed that ferroptosis could be induced in HCC via SHP-1/STAT3-mediated downregulation of MCL1 and subsequent inhibition of SLC7A11 by increased BECN1 binding.

Redox-dependent AMPK inactivation disrupts metabolic adaptation to glucose starvation in xCT-overexpressing cancer cells.

Accelerated aerobic glycolysis is a distinctive metabolic property of cancer cells that confers dependency on glucose for survival. However, the therapeutic strategies targeting this vulnerability are still inefficient and have unacceptable side effects in clinical trials. Therefore, developing biomarkers to predict therapeutic efficacy would be essential to improve the selective targeting of cancer cells. Here, we found that cell lines that are sensitive to glucose deprivation have high expression of cystine/glutamate antiporter xCT (also known as SLC7A11). We found that cystine uptake and glutamate export through xCT contributed to rapid NADPH depletion under glucose deprivation. This collapse of the redox system oxidized and inactivated AMP-activated protein kinase (AMPK), a major regulator of metabolic adaptation, resulting in a metabolic catastrophe and cell death. Although this phenomenon was prevented by pharmacological or genetic inhibition of xCT, overexpression of xCT sensitized resistant cancer cells to glucose deprivation. Taken together, these findings suggest a novel crosstalk between AMPK and xCT that links metabolism and signal transduction, and reveal a metabolic vulnerability to glucose deprivation in cancer cells expressing high levels of xCT.

Patulin disrupts SLC7A11-cystine-cysteine-GSH antioxidant system and promotes renal cell ferroptosis both in vitro and in vivo.

Patulin (PAT) is a common food-borne mycotoxin with diverse toxic effects including nephrotoxicity. The induction of oxidative stress is suggested to be a key mechanism contributed to toxicities of PAT. Reduced glutathione (GSH), a sulfhydryl-containing tripeptide, is a key reason for PAT-mediated oxidative stress. Cystine/glutamate antiporter (system xc-)-mediated cystine uptake plays a critical role in maintaining redox balance via promoting GSH biosynthesis. In this study, we addressed if GSH reduction by PAT was associated with inhibition of system xc--mediated GSH biosynthesis. Results showed that PAT significantly decreased activity of SLC7A11, a core subunit of system xc-, through activating AMPK-mediated formation of beclin1-SLC7A11 complex. Furthermore, PAT promoted ferroptosis induced by a known ferroptosis inducer RSL3 in normal renal cells, and exacerbated folic acid-induced nephrotoxicity in a mouse model of acute kidney injury. The findings of the present study provide new insights into PAT-induced kidney toxicity, and implicate that patients with ferroptosis-associated diseases maybe more susceptible to PAT.

N-Acetylcysteine Promotes Metastatic Spread of Melanoma in Mice.

Simple SummaryMalignant melanoma is a cancer derived from melanocytes, the cells that produce pigment (melanin) in the skin. It develops on the skin, but can also appear on the mucous membranes and in other locations. Melanomas are responsible for 80% of deaths related to skin cancers. In recent years, the number of cases has increased alarmingly, likely in relation to sun exposure habits. Once melanoma spreads to distant parts of the body, the 5-year survival rate is about 10%. N-acetylcysteine (NAC) is a drug with antioxidant properties, and thereby could play a role in preventing cancer. NAC is commonly used as a mucolytic in different respiratory diseases, to treat acetaminophen (Tylenol) poisoning, and is also present in different nutritional supplements. Nevertheless, the use of NAC and other antioxidants in cancer has been questioned. Here, we show that high therapeutic doses of NAC may cause metastatic spread of a malignant melanoma.N-acetylcysteine (NAC) is a direct Cys donor and a promoter of glutathione (GSH) synthesis. GSH regulates melanoma growth and NAC has been suggested to increase melanoma metastases in mice. We found that high therapeutic doses of NAC do not increase the growth of melanoma xenografts, but can cause metastatic spread and distant metastases. Nevertheless, this is not due to an antioxidant effect since NAC, in fact, increases the generation of reactive oxygen species in the growing metastatic melanoma. Trolox, an antioxidant vitamin E derivative, administered in vivo, decreased metastatic growth. Metastatic cells isolated from NAC-treated mice showed an increase in the nuclear translocation of Nrf2, as compared to controls. Nrf2, a master regulator of the antioxidant response, controls the expression of different antioxidant enzymes and of the γ-glutamylcysteine ligase (the rate-limiting step in GSH synthesis). Cystine uptake through the xCT cystine-glutamate antiporter (generating intracellular Cys) and the γ-glutamylcysteine ligase activity are key to control metastatic growth. This is associated to an increase in the utilization of L-Gln by the metastatic cells, another metastases promoter. Our results demonstrate the potential of NAC as an inducer of melanoma metastases spread, and suggest that caution should be taken when administering GSH promoters to cancer patients.

Hypersensitivity to ferroptosis in chromophobe RCC is mediated by a glutathione metabolic dependency and cystine import via solute carrier family 7 member 11

Chromophobe (Ch) renal cell carcinoma (RCC) arises from the intercalated cell in the distal nephron. There are no proven treatments for metastatic ChRCC. A distinguishing characteristic of ChRCC is strikingly high levels of reduced (GSH) and oxidized (GSSG) glutathione. Here, we demonstrate that ChRCC-derived cells exhibit higher sensitivity to ferroptotic inducers compared with clear-cell RCC. ChRCC-derived cells are critically dependent on cystine via the cystine/glutamate antiporter xCT to maintain high levels of glutathione, making them sensitive to inhibitors of cystine uptake and cyst(e)inase. Gamma-glutamyl transferase 1 (GGT1), a key enzyme in glutathione homeostasis, is markedly suppressed in ChRCC relative to normal kidney. Importantly, GGT1 overexpression inhibits the proliferation of ChRCC cells invitro and invivo, suppresses cystine uptake, and decreases levels of GSH and GSSG. Collectively, these data identify ferroptosis as a metabolic vulnerability in ChRCC, providing a potential avenue for targeted therapy for these distinctive tumors.

The Role of SLC7A11 in Cancer: Friend or Foe?

Simple SummaryStrikingly, there are many literature reports confirming that the SLC7A11 gene is closely related to the tumourigenesis, survival, proliferation, metastasis, therapeutic resistance, and other aspects of tumour cells. Meanwhile, the role of SLC7A11 in tumours is highly complex, and its pro-tumour and antitumour activities are obviously different. We summarised the biological characteristics of SLC7A11, including its structure, expression, function, regulation, and therapeutic approaches, and focused on the discussion and analysis of the possibility of SLC7A11 as a potential antitumour target, providing a theoretical basis for drug research and clinical tumour treatment.SLC7A11 controls the uptake of extracellular cystine in exchange for glutamate at a ratio of 1:1, and it is overexpressed in a variety of tumours. Accumulating evidence has shown that the expression of SLC7A11 is fine-tuned at multiple levels, and plays diverse functional and pharmacological roles in tumours, such as cellular redox homeostasis, cell growth and death, and cell metabolism. Many reports have suggested that the inhibition of SLC7A11 expression and activity is favourable for tumour therapy; thus, SLC7A11 is regarded as a potential therapeutic target. However, emerging evidence also suggests that on some occasions, the inhibition of SLC7A11 is beneficial to the survival of cancer cells, and confers the development of drug resistance. In this review, we first briefly introduce the biological properties of SLC7A11, including its structure and physiological functions, and further summarise its regulatory network and potential regulators. Then, focusing on its role in cancer, we describe the relationships of SLC7A11 with tumourigenesis, survival, proliferation, metastasis, and therapeutic resistance in more detail. Finally, since SLC7A11 has been linked to cancer through multiple approaches, we propose that its contribution and regulatory mechanism require further elucidation. Thus, more personalised therapeutic strategies should be adapted when targeting SLC7A11.

ATF4 protects against sorafenib-induced cardiotoxicity by suppressing ferroptosis

Sorafenib (SOR) is an effective chemotherapy drug for hepatocellular carcinoma, renal cell carcinoma, and differentiated thyroid carcinoma. However, a long-standing clinical issue associated with SOR use is an increased risk of cardiotoxicity, but the underlying mechanisms remain obscure. Here we report that ferroptosis of cardiomyocytes is responsible for SOR-induced cardiotoxicity. The specific ferroptosis inhibitor ferrostatin-1 and deferoxamine mesylate, an iron chelator, significantly alleviate SOR-induced cardiac damage. RNA-sequencing revealed that endoplasmic reticulum (ER) stress and the unfolded protein response were predominately activated, which might be attributed to the lipid reactive oxygen species-mediated perturbation of the ER. Activating transcription factor 4 (ATF4) is one of the most significantly up-regulated genes, knockdown of ATF4 exacerbates cardiomyocyte ferroptosis induced by SOR, while overexpression of ATF4 promotes cell survival. Mice with AAV-mediated ATF4 knockdown exhibit lipid peroxidation and more severe cardiomyopathy. Further experiments demonstrated that ATF4 exerts its protective role by elevating SLC7A11 expression, a transport subunit of system Xc−, which promotes cystine uptake and glutathione biosynthesis. The cardioprotective effect of ATF4 was diminished by SLC7A11 knockdown in cardiomyocytes subjected to SOR treatment. Taken together, these findings show that ferroptosis of cardiomyocytes is an important cause of SOR-related cardiotoxicity. ATF4 acts as a key regulator to promote cardiomyocytes survival by up-regulation of SLC7A11 and suppression of ferroptosis.

Abstract 214: Styrylbenzoic acid derivative DC10 potentiates radiotherapy by inhibiting glutathione synthesis, leading to increased oxidative stress and cancer cell death

Abstract Metastatic tumors with moderate radiosensitivity account for most cancer-related deaths, highlighting the limitations of current radiotherapy regimens. The xCT-inhibitor sulfasalazine (SAS) sensitizes cancer cells by blocking xCT-mediated cystine uptake, and thereby glutathione (GSH) synthesis protecting against radiation-induced oxidative stress. However, SAS has limited clinical potential as a radiosensitizer due to side effects and low bio-availability. Using SAS as a starting point, we previously developed synthetic xCT-inhibitors through scaffold hopping & structure optimization aided by structure-activity relationship analysis (SAR). Notably, the compound DC10 exhibited potent inhibition of GSH synthesis. In this study, we validated DC10 as a radiosensitizer in the xCT-expressing cancer cell lines A172, A375 & MCF7 in vitro & in mice harboring melanoma xenografts. After DC10 treatment, we measured 14C-cystine uptake in the cancer cell lines using liquid scintillation counting, and intracellular levels of GSH & reactive oxygen species (ROS) using luminescence assays. We performed immunoblotting for γH2AX & ATM to assess DNA damage after treatment with DC10 and radiotherapy. We then assessed the effect of adding DC10 to radiation upon cancer cell colony formation. Blood samples from mice treated with DC10 underwent biochemical analysis to assess toxicity. Finally, mice with A375 melanomas in the flank, received DC10 and radiotherapy in combination, as monotherapies or no treatment. Notably, DC10 reduced cystine uptake and GSH synthesis & increased ROS levels in a dose-dependent manner. Furthermore, DC10 interacted synergistically with radiation to increase DNA damage & reduce colony formation. Mice receiving DC10 were clinically unaffected, whereas blood samples analyses to assess bone marrow suppression, liver or kidney toxicity revealed no significant differences between mice & untreated controls. Importantly, DC10 potentiated the anti-tumor efficacy of radiation in mice with melanoma xenografts. We conclude that DC10 acts as a radiosensitizer, is well tolerated, & mediates its effect by inhibiting cystine uptake, leading to GSH depletion and increased oxidative stress. Thus, our findings demonstrate the feasibility of using synthetic xCT-inhibitors to overcome radioresistance. The expression of xCT in multiple tumor types further implies it represents a target generic to cancer rather than confined to tumor subtypes. Hence, this therapeutic concept could be applicable to a wide range of radioresistant malignancies. Citation Format: Shahin Sarowar, Davide Cirillo, Pablo Jativa, Mette H. Nilsen, Sarah-Muheha A. Otragane, Jan I. Heggdal, Frode Selheim, Valentín Ceña, Hans-René Bjørsvik, Per Øyvind Enger. Styrylbenzoic acid derivative DC10 potentiates radiotherapy by inhibiting glutathione synthesis, leading to increased oxidative stress and cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 214.

Melatonin modulates Nrf2 activity to protect porcine pre-pubertal Sertoli cells from the abnormal H2 O2 generation and reductive stress effects of cadmium.

Melatonin (MLT) is a cytoprotective agent holding potential to prevent cadmium (Cd) toxicity and its impact in testicular function and fertility. In this study, we explored such potential in porcine pre‐pubertal Sertoli cells (SCs). Cd toxicity resulted in impaired SC viability and function, abnormal cellular H2O2 generation and efflux, and induction of reductive stress by the upregulation of Nrf2 expression and activity, cystine uptake and glutathione biosynthesis, glutathione‐S‐transferase P (GSTP) expression, and protein glutathionylation inhibition. Cd toxicity also stimulated the activity of cellular kinases (MAPK‐ERK1/2 and Akt) and NFkB transcription factor, and cJun expression was increased. MLT produced a potent cytoprotective effect when co‐administered with Cd to SCs; its efficacy and the molecular mechanism behind its cytoprotective function varied according to Cd concentrations. However, a significant restoration of cell viability and function, and of H2O2 levels, was observed both at 5 and 10 μM Cd. Mechanistically, these effects of MLT were associated with a significant reduction of the Cd‐induced activation of Nrf2 and GSTP expression at all Cd concentrations. CAT and MAPK‐ERK1/2 activity upregulation was associated with these effects at 5 μM Cd, whereas glutathione biosynthesis and efflux were involved at 10 μM Cd together with an increased expression of the cystine transporter xCT, of cJun and Akt and NFkB activity. MLT protects SCs from Cd toxicity reducing its H2O2 generation and reductive stress effects. A reduced activity of Nrf2 and the modulation of other molecular players of MLT signaling, provide a mechanistic rational for the cytoprotective effect of this molecule in SCs.

Cystine uptake inhibition potentiates front-line therapies in acute myeloid leukemia

By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.

CEMIP promotes extracellular matrix‐detached prostate cancer cell survival by inhibiting ferroptosis

Cells detached from the extracellular matrix (ECM) can trigger different modes of cell death, and the survival of ECM‐detached cells is one of the prerequisites for the metastatic cascade. Ferroptosis, a form of iron‐dependent programmed cell death, has recently been found to be involved in matrix‐detached cancer cells. However, the molecular mechanisms by which ECM‐detached cells escape ferroptosis are not fully understood. Here, we observed that cell migration‐inducing protein (CEMIP) upregulation facilitates ferroptosis resistance during ECM detachment by promoting cystine uptake in prostate cancer (PCa) cells. Meanwhile, silencing CEMIP causes it to lose its ability to promote cystine uptake and inhibit ferroptosis. Mechanistically, the interaction of CEMIP with inositol 1,4,5‐trisphosphate receptor type 3 (ITPR3) modulates calcium ion (Ca2+) leakage from the endoplasmic reticulum, activating calcium/calmodulin‐dependent protein kinase II (CaMKII), which further facilitates nuclear factor erythroid 2‐related factor 2 (NRF2) phosphorylation and nuclear localization, leading to elevated transcription of solute carrier family 7 member 11 (SLC7A11), a glutamate/cystine antiporter, in PCa cells. Our findings delineate a novel role of CEMIP in ferroptosis resistance during ECM detachment and provide new insights into therapeutic strategies for metastatic PCa.

MYCN mediates cysteine addiction and sensitizes neuroblastoma to ferroptosis

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

Synergistic Effect of Erastin Combined with Nutlin-3 on Vestibular Schwannoma Cells as p53 Modulates Erastin-Induced Ferroptosis Response.

Vestibular schwannoma (VS) is a rare neurotology neoplasm that results in partial neurological defects. As we know, a comprehensive understanding of basic mechanisms and targeted therapy is vital for disease management. In VS, p53 has been proved to suppress tumor progression via a cooperative with the key protein, merlin, as well as regulation of the cell cycle. However, there are more potential mechanisms of p53 in VS needed to exploit. First, via genome-wide RNA expression analysis, we identified differentially expressed genes in VS compared with normal nerves, and then, bioinformatics analyses were used to analyze these differential expression data and suggested a high level of enrichment of cysteine and glutathione metabolism pathways in VS. Meanwhile, we observed a downregulation of SLC7A11/xCT, a component of the cystine/glutamate antiporter (also known as system xc−) involved in cystine uptake. Next, for a deeper study, our group extracted tumor cells from vestibular schwannoma tissues and established two immortalized cell lines named JEI-001 and JEI-002. Secondly, in our established cells, we demonstrated that ferroptosis participated in erastin-induced growth inhibition. As a novel cell death process, ferroptosis driven by iron-mediated lipid reactive oxygen species (lipid ROS), as well as cysteine and glutathione metabolism. Furthermore, ferroptosis contributes to the inhibitory effects of tumor suppressor p53. Here, we show that p53 sensitizes schwannoma cells to ferroptosis by repressing expression of SLC7A11/xCT. Finally, erastin combined with Nutlin-3, which s to p53 activation, triggered antitumor effects of ferroptosis on the growth of schwannoma cells in vitro. These findings present potential mechanism of p53 in schwannomas and raise the possibility of treatment strategies directed against this pathogenesis.

Cysteine dependence of Lactobacillus iners is a potential therapeutic target for vaginal microbiota modulation.

Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from >300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

The Role of Cystine/Glutamate Antiporter SLC7A11/xCT in the Pathophysiology of Cancer.

SLC7A11/xCT is an antiporter that mediates the uptake of extracellular cystine in exchange for glutamate. Cystine is reduced to cysteine, which is a rate-limiting precursor in glutathione synthesis; a process that protects cells from oxidative stress and is, therefore, critical to cell growth, proliferation, and metabolism. SLC7A11 is expressed in different tissues and plays diverse functional roles in the pathophysiology of various diseases, including cancer, by regulating the processes of redox homeostasis, metabolic flexibility/nutrient dependency, immune system function, and ferroptosis. SLC7A11 expression is associated with poor prognosis and drug resistance in cancer and, therefore, represents an important therapeutic target. In this review, we discuss the molecular functions of SLC7A11 in normal versus diseased tissues, with a special focus on how it regulates gastrointestinal cancers. Further, we summarize current therapeutic strategies targeting SLC7A11 as well as novel avenues for treatment.

MYCN upregulates the transsulfuration pathway to suppress the ferroptotic vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis is an iron-dependent, oxidative form of cell death that is countered mainly by glutathione peroxidase 4 (GPX4) and the production of glutathione (GSH), which is formed from cysteine. The identification of the cancers that may benefit from pharmacological ferroptotic induction is just emerging. We recently demonstrated that inducing ferroptosis genetically or pharmacologically in MYCN-amplified neuroblastoma (NB) is a novel and effective way to kill these cells. MYCN increases iron metabolism and subsequent hydroxyl radicals through increased expression of the transferrin receptor 1 (TfR1) and low levels of the ferroportin receptor. To counter increased hydroxyl radicals, MYCN binds to the promoter of SLC3A2 (solute carrier family 3 member 2). SLC3A2 is a subunit of system Xc-, which is the cysteine-glutamate antiporter that exports glutamate and imports cystine. Cystine is converted to cysteine intracellularly. Here, we investigated other ways MYCN may increase cysteine levels. By performing metabolomics in a syngeneic NB cell line either expressing MYCN or GFP, we demonstrate that the transsulfuration pathway is activated by MYCN. Furthermore, we demonstrate that MYCN-amplified NB cell lines and tumors have higher levels of cystathionine beta-synthase (CBS), the rate-limiting enzyme in transsulfuration, which leads to higher levels of the thioether cystathionine (R-S-(2-amino-2-carboxyethyl)-l-homocysteine). In addition, MYCN-amplified NB tumors have high levels of methylthioadenosine phosphorylase (MTAP), an enzyme that helps salvage methionine following polyamine metabolism. MYCN directly binds to the promoter of MTAP. We propose that MYCN orchestrates both enhanced cystine uptake and enhanced activity of the transsulfuration pathway to counteract increased reactive oxygen species (ROS) from iron-induced Fenton reactions, ultimately contributing to a ferroptosis vulnerability in MYCN-amplified neuroblastoma.

Expression of gamma-glutamyltransferase 1 in glioblastoma cells confers resistance to cystine deprivation–induced ferroptosis

Ferroptosis is an iron-dependent mode of cell death caused by excessive oxidative damage to lipids. Lipid peroxidation is normally suppressed by glutathione peroxidase 4, which requires reduced glutathione. Cystine is a major resource for glutathione synthesis, especially in cancer cells. Therefore, cystine deprivation or inhibition of cystine uptake promotes ferroptosis in cancer cells. However, the roles of other molecules involved in cysteine deprivation–induced ferroptosis are unexplored. We report here that the expression of gamma-glutamyltransferase 1 (GGT1), an enzyme that cleaves extracellular glutathione, determines the sensitivity of glioblastoma cells to cystine deprivation–induced ferroptosis at high cell density (HD). In glioblastoma cells expressing GGT1, pharmacological inhibition or deletion of GGT1 suppressed the cell density–induced increase in intracellular glutathione levels and cell viability under cystine deprivation, which were restored by the addition of cysteinylglycine, the GGT product of glutathione cleavage. On the other hand, cystine deprivation induced glutathione depletion and ferroptosis in GGT1-deficient glioblastoma cells even at an HD. Exogenous expression of GGT1 in GGT1-deficient glioblastoma cells inhibited cystine deprivation–induced glutathione depletion and ferroptosis at an HD. This suggests that GGT1 plays an important role in glioblastoma cell survival under cystine-limited and HD conditions. We conclude that combining GGT inhibitors with ferroptosis inducers may provide an effective therapeutic approach for treating glioblastoma.