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Iron is an essential element for life owing to its ability to participate in a diverse array of oxidation-reduction reactions. However, misregulation of iron-dependent redox cycling can also produce oxidative stress, contributing to cell growth, proliferation, and death pathways underlying aging, cancer, neurodegeneration, and metabolic diseases. Fluorescent probes that selectively monitor loosely bound Fe(II) ions, termed the labile iron pool, are potentially powerful tools for studies of this metal nutrient; however, the dynamic spatiotemporal nature and potent fluorescence quenching capacity of these bioavailable metal stores pose challenges for their detection. Here, we report a tandem activity-based sensing and labeling strategy that enables imaging of labile iron pools in live cells through enhancement in cellular retention. Iron green-1 fluoromethyl (IG1-FM) reacts selectively with Fe(II) using an endoperoxide trigger to release a quinone methide dye for subsequent attachment to proximal biological nucleophiles, providing a permanent fluorescent stain at sites of elevated labile iron. IG1-FM imaging reveals that degradation of the major iron storage protein ferritin through ferritinophagy expands the labile iron pool, while activation of nuclear factor-erythroid 2-related factor 2 (NRF2) antioxidant response elements (AREs) depletes it. We further show that lung cancer cells with heightened NRF2 activation, and thus lower basal labile iron, have reduced viability when treated with an iron chelator. By connecting labile iron pools and NRF2-ARE activity to a druggable metal-dependent vulnerability in cancer, this work provides a starting point for broader investigations into the roles of transition metal and antioxidant signaling pathways in health and disease.

Copper is an essential metal nutrient for life that often relies on redox cycling between Cu(I) and Cu(II) oxidation states to fulfill its physiological roles, but alterations in cellular redox status can lead to imbalances in copper homeostasis that contribute to cancer and other metalloplasias with metal-dependent disease vulnerabilities. Copper-responsive fluorescent probes offer powerful tools to study labile copper pools, but most of these reagents target Cu(I), with limited methods for monitoring Cu(II) owing to its potent fluorescence quenching properties. Here, we report an activity-based sensing strategy for turn-on, oxidation state-specific detection of Cu(II) through metal-directed acyl imidazole chemistry. Cu(II) binding to a metal and oxidation state-specific receptor that accommodates the harder Lewis acidity of Cu(II) relative to Cu(I) activates the pendant dye for reaction with proximal biological nucleophiles and concomitant metal ion release, thus avoiding fluorescence quenching. Copper-directed acyl imidazole 649 for Cu(II) (CD649.2) provides foundational information on the existence and regulation of labile Cu(II) pools, including identifying divalent metal transporter 1 (DMT1) as a Cu(II) importer, labile Cu(II) increases in response to oxidative stress induced by depleting total glutathione levels, and reciprocal increases in labile Cu(II) accompanied by decreases in labile Cu(I) induced by oncogenic mutations that promote oxidative stress.

The non-essential amino acid serine is a critical nutrient for cancer cells due to its diverse biosynthetic functions. While some tumors can synthesize serine de novo , others are auxotrophic and therefore reliant on serine uptake. Importantly, despite several transporters being known to be capable of transporting serine, the transporters that mediate serine uptake in cancer cells are not known. Here, we characterize the amino acid transporter ASCT2 (SLC1A5) as a major contributor to serine uptake in cancer cells. ASCT2 is well known as a glutamine transporter in cancer, and our work demonstrates that serine and glutamine compete for uptake through ASCT2. We further show that ASCT2-mediated serine uptake is essential for purine nucleotide biosynthesis and that estrogen receptor α (ERα) promotes serine uptake by directly activating SLC1A5 transcription. Collectively, our work defines an additional important role for ASCT2 as a serine transporter in cancer and evaluates ASCT2 as a potential therapeutic target. [Display omitted] • ASCT2 contributes to serine uptake in cancer cells • Serine and glutamine compete for uptake via ASCT2 • ERα promotes serine uptake by directly activating SLC1A5 transcription • Loss of ASCT2 combined with dietary serine starvation shrinks xenograft tumors Despite significant interest in targeting serine metabolism for cancer therapy, the transporters that mediate serine uptake in cancer cells are not known. Conger et al. describe the amino acid transporter ASCT2 as a major contributor to serine uptake in cancer cells that is required for tumor growth in low-serine conditions. [ABSTRACT FROM AUTHOR]

Cancer cells reprogram their metabolism to promote growth and proliferation. The genetic evidence pointing to the importance of the amino acid serine in tumorigenesis is striking. The gene encoding the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first committed step of serine biosynthesis, is overexpressed in tumors and cancer cell lines via focal amplification and nuclear factor erythroid-2-related factor 2 (NRF2)-mediated up-regulation. PHGDH-overexpressing cells are exquisitely sensitive to genetic ablation of the pathway. Here, we report the discovery of a selective small molecule inhibitor of PHGDH, CBR-5884, identified by screening a library of 800,000 drug-like compounds. CBR-5884 inhibited de novo serine synthesis in cancer cells and was selectively toxic to cancer cell lines with high serine biosynthetic activity. Biochemical characterization of the inhibitor revealed that it was a noncompetitive inhibitor that showed a time-dependent onset of inhibition and disrupted the oligomerization state of PHGDH. The identification of a small molecule inhibitor of PHGDH not only enables thorough preclinical evaluation of PHGDH as a target in cancers, but also provides a tool with which to study serine metabolism. [ABSTRACT FROM AUTHOR]

Simple Summary: The stress-responsive transcription factor NRF2 (nuclear factor-erythroid 2 p45-related factor 2) directs cellular metabolic processes that can have diverse effects in the context of cancer. This review addresses how NRF2 and its negative regulator KEAP1 (Kelch-like ECH-associated protein 1) collectively modulate and respond to metabolism. We highlight NRF2-regulated processes relevant to the antioxidant system, cellular proliferation, and survival, including metabolism of amino acids, lipids, NADPH (reduced nicotinamide adenine dinucleotide phosphate), iron, and heme. We also review the stabilization of NRF2 by electrophiles, metabolites, and autophagy. Finally, we discuss topics that warrant further investigation into the KEAP1/NRF2 pathway's role in tumor progression. The transcription factor NRF2 (nuclear factor-erythroid 2 p45-related factor 2 or NFE2L2) plays a critical role in response to cellular stress. Following an oxidative insult, NRF2 orchestrates an antioxidant program, leading to increased glutathione levels and decreased reactive oxygen species (ROS). Mounting evidence now implicates the ability of NRF2 to modulate metabolic processes, particularly those at the interface between antioxidant processes and cellular proliferation. Notably, NRF2 regulates the pentose phosphate pathway, NADPH production, glutaminolysis, lipid and amino acid metabolism, many of which are hijacked by cancer cells to promote proliferation and survival. Moreover, deregulation of metabolic processes in both normal and cancer-based physiology can stabilize NRF2. We will discuss how perturbation of metabolic pathways, including the tricarboxylic acid (TCA) cycle, glycolysis, and autophagy can lead to NRF2 stabilization, and how NRF2-regulated metabolism helps cells deal with these metabolic stresses. Finally, we will discuss how the negative regulator of NRF2, Kelch-like ECH-associated protein 1 (KEAP1), may play a role in metabolism through NRF2 transcription-independent mechanisms. Collectively, this review will address the interplay between the NRF2/KEAP1 complex and metabolic processes. [ABSTRACT FROM AUTHOR]