Ferroptosis inhibition by oleic acid mitigates iron-overload-induced injury.

Iron overload, characterized by accumulation of iron in tissues, induces a multiorgan toxicity whose mechanisms are not fully understood. Using cultured cell lines, Caenorhabditis elegans, and mice, we found that ferroptosis occurs in the context of iron-overload-mediated damage. Exogenous oleic acid protected against iron-overload-toxicity in cell culture and Caenorhabditis elegans by suppressing ferroptosis. In mice, oleic acid protected against FAC-induced liver lipid peroxidation and damage. Oleic acid changed the cellular lipid composition, characterized by decreased levels of polyunsaturated fatty acyl phospholipids and decreased levels of ether-linked phospholipids. The protective effect of oleic acid in cells was attenuated by GW6471 (PPAR-α antagonist), as well as in Caenorhabditis elegans lacking the nuclear hormone receptor NHR-49 (a PPAR-α functional homologue). These results highlight ferroptosis as a driver of iron-overload-mediated damage, which is inhibited by oleic acid. This monounsaturated fatty acid represents a potential therapeutic approach to mitigating organ damage in iron overload individuals.

Probing the Cytotoxic Signaling Induced by Eupenifeldin in Ovarian Cancer Models.

High-grade serous ovarian cancer (HGSOC) is the most common and lethal ovarian cancer histotype. Lack of early detection methods, limited therapeutic agents, and low 5-year survival rate reflect the urgent need to develop new therapies. Eupenifeldin, a bistropolone, originally isolated from Eupenicillium brefeldianum, is a cytotoxic fungal metabolite. In three HSGOC cell lines (OVCAR3, OVCAR5, OVCAR8), eupenifeldin was found to have an IC50 value less than 10 nM, while 10 times higher concentrations were required for cytotoxicity in nontumorigenic fallopian tube secretory epithelial cell lines (FTSEC). An in vivo hollow fiber assay showed significant cytotoxicity in OVCAR3. Eupenifeldin significantly increased Annexin V staining in OVCAR3 and -8, but not OVCAR5. Eupenifeldin activated caspases 3/7 in OVCAR3, OVCAR5, and OVCAR8; however, cleaved PARP was only detected in OVCAR3. Quantitative proteomics performed on OVCAR3 implicated ferroptosis as the most enriched cell death pathway. However, validation experiments did not support ferroptosis as part of the cytotoxic mechanism of eupenifeldin. Autophagic flux and LC3B puncta assays found that eupenifeldin displayed weak autophagic induction in OVCAR3. Inhibition of autophagy by cotreatment with bafilomycin reduced the toxicity of eupenifeldin, supporting the idea that induction of autophagy contributes to the cytotoxic mechanism of eupenifeldin.

Identification of essential sites of lipid peroxidation in ferroptosis.

Ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, provides a potential treatment avenue for drug-resistant cancers and may play a role in the pathology of some degenerative diseases. Identifying the subcellular membranes essential for ferroptosis and the sequence of their peroxidation will illuminate drug discovery strategies and ferroptosis-relevant disease mechanisms. In this study, we employed fluorescence and stimulated Raman scattering imaging to examine the structure-activity-distribution relationship of ferroptosis-modulating compounds. We found that, although lipid peroxidation in various subcellular membranes can induce ferroptosis, the endoplasmic reticulum (ER) membrane is a key site of lipid peroxidation. Our results suggest an ordered progression model of membrane peroxidation during ferroptosis that accumulates initially in the ER membrane and later in the plasma membrane. Thus, the design of ER-targeted inhibitors and inducers of ferroptosis may be used to optimally control the dynamics of lipid peroxidation in cells undergoing ferroptosis.

Vitamin K: A new guardian against ferroptosis.

The dietary factor vitamin K has been found to protect against ferroptosis, a form of cell death driven by lipid peroxidation. This reveals new dietary links to cancers and degenerative conditions and a key factor involved in warfarin poisoning.

Mesenchymal subtype neuroblastomas are addicted to TGF-ßR2/HMGCR-driven protein geranylgeranylation.

The identification of targeted agents with high therapeutic index is a major challenge for cancer drug discovery. We found that screening chemical libraries across neuroblastoma (NBL) tumor subtypes for selectively-lethal compounds revealed metabolic dependencies that defined each subtype. Bioactive compounds were screened across cell models of mesenchymal (MESN) and MYCN-amplified (MYCNA) NBL subtypes, which revealed the mevalonate and folate biosynthetic pathways as MESN-selective dependencies. Treatment with lovastatin, a mevalonate biosynthesis inhibitor, selectively inhibited protein prenylation and induced apoptosis in MESN cells, while having little effect in MYCNA lines. Statin sensitivity was driven by HMGCR expression, the rate-limiting enzyme for cholesterol synthesis, which correlated with statin sensitivity across NBL cell lines, thus providing a drug sensitivity biomarker. Comparing expression profiles from sensitive and resistant cell lines revealed a TGFBR2 signaling axis that regulates HMGCR, defining an actionable addiction in that leads to MESN-subtype-dependent apoptotic cell death.

Cysteine depletion induces pancreatic tumor ferroptosis in mice.

Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC - is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC - subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.

The development of the concept of ferroptosis.

The term ferroptosis was coined in 2012 to describe an iron-dependent regulated form of cell death caused by the accumulation of lipid-based reactive oxygen species; this type of cell death was found to have molecular characteristics distinct from other forms of regulated cell death. Features of ferroptosis have been observed periodically over the last several decades, but these molecular features were not recognized as evidence of a distinct form of cell death until recently. Here, we describe the history of observations consistent with the current definition of ferroptosis, as well as the advances that contributed to the emergence of the concept of ferroptosis. We also discuss recent implications and applications of manipulations of the ferroptotic death pathway.

Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling.

The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N-glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N-glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N-glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy-D-glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.

Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-ß.

Transforming growth factor-ß (TGF-ß) ligands activate Smad-mediated and noncanonical signaling pathways in a cell context-dependent manner. Localization of signaling receptors to distinct membrane domains is a potential source of signaling output diversity. The tumor suppressor/endocytic adaptor protein disabled-2 (Dab2) was proposed as a modulator of TGF-ß signaling. However, the molecular mechanism(s) involved in the regulation of TGF-ß signaling by Dab2 were not known. Here we investigate these issues by combining biophysical studies of the lateral mobility and endocytosis of the type I TGF-ß receptor (TßRI) with TGF-ß phosphoprotein signaling assays. Our findings demonstrate that Dab2 interacts with TßRI to restrict its lateral diffusion at the plasma membrane and enhance its clathrin-mediated endocytosis. Small interfering RNA-mediated knockdown of Dab2 or Dab2 overexpression shows that Dab2 negatively regulates TGF-ß-induced c-Jun N-terminal kinase (JNK) activation, whereas activation of the Smad pathway is unaffected. Moreover, activation of JNK by TGF-ß in the absence of Dab2 is disrupted by cholesterol depletion. These data support a model in which Dab2 regulates the domain localization of TßRI in the membrane, balancing TGF-ß signaling via the Smad and JNK pathways.

Differential regulation of Smad3 and of the type II transforming growth factor-ß receptor in mitosis: implications for signaling.

The response to transforming growth factor-ß (TGF-ß) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-ß receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-ß stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-ß receptor (TßRII). Moreover, following the stimulation of mitotic cells with TGF-ß, the proteasome-mediated attenuation of TGF-ß receptor activity, the degradation and clearance of TßRII from the plasma membrane, and the clearance of the TGF-ß ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-ß receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-ß stimuli in mitosis.