Biology subjects

Ward, N. P.

Publications and source records attributed to Ward, N. P..

7 recordsLinked to original sources

bioRxiv2026

Cancer cells acquire cysteine directly from a reduced tumor microenvironment

The metabolic environment of a tumor is shaped by the nutrients available in its interstitial fluid, yet the redox composition of that fluid is poorly defined. Measuring sulfur metabolites in the interstitial fluid of tumors, matched healthy organs, and serum across three mouse models of cancer, we find that the tumor microenvironment (TME) is a chemically reduced compartment in which free cysteine predominates over its oxidized form, cystine. This is especially pronounced in the lung TME, where cystine is nearly absent. We further show that lung cancer cells take up cysteine and cystine with comparable efficiency and proliferate on cysteine alone, and that the abundant glutathione in the TME helps keep cysteine reduced through disulfide exchange. In a medium formulated to match the sulfur composition of the lung tumor interstitial fluid, cells become independent of cystine import through xCT. Cells cultured in this medium were resistant to glutaminase inhibitors, oxidants and ferroptosis inducers, and better maintained NADPH levels under oxidative challenge. These findings suggest that standard culture conditions overestimate the dependence of cancer cells on cystine uptake and their vulnerability to oxidative stress-targeting therapies.

cancer biology↗

bioRxiv2024

Catabolism of extracellular glutathione supplies amino acids to support tumor growth

Restricting amino acids from tumors is an emerging therapeutic strategy with significant promise. While typically considered an intracellular antioxidant with tumor-promoting capabilities, glutathione (GSH) is a tripeptide of cysteine, glutamate, and glycine that can be catabolized, yielding amino acids. The extent to which GSH-derived amino acids are essential to cancers is unclear. Here, we find that GSH catabolism promotes tumor growth. We show that depletion of intracellular GSH does not perturb tumor growth, and extracellular GSH is highly abundant in the tumor microenvironment, highlighting the potential importance of GSH outside of tumors. We find supplementation with GSH can rescue cancer cell survival and growth in cystine-deficient conditions, and this rescue is dependent on the catabolic activity of {gamma}-glutamyltransferases (GGTs). Finally, pharmacologic targeting of GGTs activity prevents the breakdown of circulating GSH, lowers tumor cysteine levels, and slows tumor growth. Our findings indicate a non-canonical role for GSH in supporting tumors by acting as a reservoir of amino acids. Depriving tumors of extracellular GSH or inhibiting its breakdown is potentially a therapeutically tractable approach for patients with cancer. Further, these findings change our view of GSH and how amino acids, including cysteine, are supplied to cells.

cancer biology↗

bioRxiv2023

Imaging the master regulator of the antioxidant response in non-small cell lung cancer with positron emission tomography

Mutations in the NRF2-KEAP1 pathway are common in non-small cell lung cancer (NSCLC) and confer broad-spectrum therapeutic resistance, leading to poor outcomes. The cystine/glutamate antiporter, system xc-, is one of the >200 cytoprotective proteins controlled by NRF2, which can be non-invasively imaged by (S)-4-(3-18F-fluoropropyl)--glutamate ([18F]FSPG) positron emission tomography (PET). Through genetic and pharmacologic manipulation, we show that [18F]FSPG provides a sensitive and specific marker of NRF2 activation in advanced preclinical models of NSCLC. We validate imaging readouts with metabolomic measurements of system xc- activity and their coupling to intracellular glutathione concentration. A redox gene signature was measured in patients from the TRACERx 421 cohort, suggesting an opportunity for patient stratification prior to imaging. Furthermore, we reveal that system xc- is a metabolic vulnerability that can be therapeutically targeted for sustained tumour growth suppression in aggressive NSCLC. Our results establish [18F]FSPG as predictive marker of therapy resistance in NSCLC and provide the basis for the clinical evaluation of both imaging and therapeutic agents that target this important antioxidant pathway.

cancer biology↗

bioRxiv2023

Glutathione supports lipid abundance in vivo

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the livers balance of redox buffering and triglyceride production.

biochemistry↗

bioRxiv2022

Mitochondrial Function is Preserved Under Cysteine Starvation via Glutathione Catabolism in NSCLC

Cysteine metabolism occurs across cellular compartments to support diverse biological functions and prevent the induction of ferroptosis. Though the disruption of cytosolic cysteine metabolism is implicated in this form of cell death, it is unknown whether the substantial cysteine metabolism resident within the mitochondria is similarly pertinent to ferroptosis. Here, we show that despite the rapid depletion of intracellular cysteine upon loss of extracellular cystine, cysteine-dependent synthesis of Fe-S clusters persists in the mitochondria of lung cancer cells. This promotes a retention of respiratory function and a maintenance of the mitochondrial redox state. Under these limiting conditions, we find that mitochondrial glutathione sustains the function of the Fe-S proteins critical to oxidative metabolism. This is achieved through CHAC1 catabolism of the cysteine-containing tripeptide within the mitochondrial matrix. We find that disrupting Fe-S cluster synthesis under cysteine restriction protects against the induction of ferroptosis, suggesting that the preservation of mitochondrial function is antagonistic to survival under starved conditions. Overall, our findings implicate mitochondrial cysteine metabolism in the induction of ferroptosis and reveal a novel mechanism of mitochondrial resilience in response to nutrient stress.

cancer biology↗

bioRxiv2022

A CRISPR screen identifies redox vulnerabilities for KEAP1/NRF2 mutant non-small cell lung cancer

The redox regulator NRF2 is hyperactivated in a large percentage of non-small cell lung cancer (NSCLC) cases, which is associated with chemotherapy and radiation resistance. To identify redox vulnerabilities for KEAP1/NRF2 mutant NSCLC, we conducted a CRISPR-Cas9-based negative selection screen for antioxidant enzyme genes whose loss sensitized cells to sub-lethal concentrations of the superoxide (O2*-)-generating drug {beta}-Lapachone. While our screen identified expected hits in the pentose phosphate pathway, the thioredoxin-dependent antioxidant system, and glutathione reductase, we also identified the mitochondrial superoxide dismutase 2 (SOD2) as one of the top hits. Surprisingly, {beta}-Lapachone did not generate mitochondrial O2*- but rather SOD2 loss enhanced the efficacy of {beta}-Lapachone due to loss of iron-sulfur protein function, loss of mitochondrial ATP maintenance and deficient NADPH production. Importantly, inhibition of mitochondrial electron transport activity sensitized to {beta}-Lapachone, demonstrating these effects may be translated to increase ROS sensitivity therapeutically.

cancer biology↗

bioRxiv2019

NNT Regulates Mitochondrial Metabolism in NSCLC Through Maintenance of Fe-S Protein Function

Human lung tumors exhibit robust and complex mitochondrial metabolism, likely precipitated by the highly oxygenated nature of pulmonary tissue. As ROS generation is a byproduct of this metabolism, reducing power in the form of nicotinamide adenine dinucleotide phosphate (NADPH) is required to mitigate oxidative stress in response to this heightened mitochondrial activity. Nicotinamide nucleotide transhydrogenase (NNT) is known to sustain mitochondrial antioxidant capacity through the generation of NADPH, however its function in non-small cell lung cancer (NSCLC) has not been established. We found that NNT expression significantly enhances tumor formation and aggressiveness in mouse models of lung tumor initiation and progression. We further show that NNT loss elicits mitochondrial dysfunction independent of substantial increases in oxidative stress, but rather marked by the diminished activities of proteins dependent on resident iron-sulfur clusters. These defects were associated with both NADPH availability and ROS accumulation, suggesting that NNT serves a specific role in mitigating the oxidation of these critical protein cofactors.

cancer biology↗