Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease.

Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer's, Huntington's, and Parkinson's diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.

TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation.

Adaptation of the endoplasmic reticulum (ER) pathway for MHC class I (MHC-I) presentation in dendritic cells enables cross-presentation of peptides derived from phagocytosed microbes, infected cells, or tumor cells to CD8 T cells. How these peptides intersect with MHC-I molecules remains poorly understood. Here, we show that MHC-I selectively accumulate within phagosomes carrying microbial components, which engage Toll-like receptor (TLR) signaling. Although cross-presentation requires Sec22b-mediated phagosomal recruitment of the peptide loading complex from the ER-Golgi intermediate compartment (ERGIC), this step is independent of TLR signaling and does not deliver MHC-I. Instead, MHC-I are recruited from an endosomal recycling compartment (ERC), which is marked by Rab11a, VAMP3/cellubrevin, and VAMP8/endobrevin and holds large reserves of MHC-I. While Rab11a activity stocks ERC stores with MHC-I, MyD88-dependent TLR signals drive IκB-kinase (IKK)2-mediated phosphorylation of phagosome-associated SNAP23. Phospho-SNAP23 stabilizes SNARE complexes orchestrating ERC-phagosome fusion, enrichment of phagosomes with ERC-derived MHC-I, and subsequent cross-presentation during infection.

Leucine retention in lysosomes is regulated by starvation.

Cells acquire essential nutrients from the environment and utilize adaptive mechanisms to survive when nutrients are scarce. How nutrients are trafficked and compartmentalized within cells and whether they are stored in response to stress remain poorly understood. Here, we investigate amino acid trafficking and uncover evidence for the lysosomal transit of numerous essential amino acids. We find that starvation induces the lysosomal retention of leucine in a manner requiring RAG-GTPases and the lysosomal protein complex Ragulator, but that this process occurs independently of mechanistic target of rapamycin complex 1 activity. We further find that stored leucine is utilized in protein synthesis and that inhibition of protein synthesis releases lysosomal stores. These findings identify a regulated starvation response that involves the lysosomal storage of leucine.

Amino acids and mechanistic target of rapamycin regulate the fate of live engulfed cells.

Metabolic stress contributes to the regulation of cell death in normal and diseased tissues. While different forms of cell death are known to be regulated by metabolic stress, how the cell engulfment and killing mechanism entosis is regulated is not well understood. Here we find that the death of entotic cells is regulated by the presence of amino acids and activity of the mechanistic target of rapamycin (mTOR). Amino acid withdrawal or mTOR inhibition induces apoptosis of engulfed cells and blocks entotic cell death that is associated with the lipidation of the autophagy protein microtubule-associated protein light chain 3 (LC3) to entotic vacuoles. Two other live cell engulfment programs, homotypic cell cannibalism (HoCC) and anti-CD47 antibody-mediated phagocytosis, known as phagoptosis, also undergo a similar vacuole maturation sequence involving LC3 lipidation and lysosome fusion, but only HoCC involves mTOR-dependent regulation of vacuole maturation and engulfed cell death similar to entosis. We further find that the regulation of cell death by mTOR is independent of autophagy activation and instead involves the 4E-BP1/2 proteins that are known regulators of mRNA translation. Depletion of 4E-BP1/2 proteins can restore the mTOR-regulated changes of entotic death and apoptosis rates of engulfed cells. These results identify amino acid signaling and the mTOR-4E-BP1/2 pathway as an upstream regulation mechanism for the fate of live engulfed cells formed by entosis and HoCC.

Genetic and clinical correlates of entosis in pancreatic ductal adenocarcinoma.

Entosis is a type of regulated cell death that promotes cancer cell competition. Though several studies have revealed the molecular mechanisms that govern entosis, the clinical and genetic correlates of entosis in human tumors is less well understood. Here we reviewed entotic cell-in-cell (CIC) patterns in a large single institution sequencing cohort (MSK IMPACT clinical sequencing cohort) of more than 1600 human pancreatic ductal adenocarcinoma (PDAC) samples to identify the genetic and clinical correlates of this cellular feature. After case selection, 516 conventional PDACs and 21 ASCs entered this study and ~45,000 HPFs (median 80 HPFs per sample) were reviewed; 549 entotic-CICs were detected through our cohort. We observed that entotic-CIC occurred more frequently in liver metastasis compared with primary in PDAC. Moreover, poorly differentiated adenocarcinoma or adenosquamous carcinoma had more entotic-CIC than well or moderately differentiated adenocarcinoma. With respect to genetic features TP53 mutations, KRAS amplification, and MYC amplification were significantly associated with entosis in PDAC tissues. From a clinical standpoint entotic CICs were independently associated with a poor prognosis by multivariate Cox regression analysis when considering all cases or primary PDACs specifically. These results provide a contextual basis for understanding entosis in PDAC, a highly aggressive cancer for which molecular insights are needed to improve survival.

The cell biology of cell-in-cell structures.

For decades, authors have described unusual cell structures, referred to as cell-in-cell structures, in which whole cells are found in the cytoplasm of other cells. One well-characterized process that results in the transient appearance of such structures is the engulfment of apoptotic cells by phagocytosis. However, many other types of cell-in-cell structure have been described that involve viable non-apoptotic cells. Some of these structures seem to form by the invasion of one cell into another, rather than by engulfment. The mechanisms of cell-in-cell formation and the possible physiological roles of these processes will be discussed.

After-Death Functions of Cell Death.

Cell death can occur through numerous regulated mechanisms, from apoptosis to necrosis, entosis, and others. Each has a distinct mode of regulation and effect on tissue homeostasis. While the elimination of individual cells is typically considered the relevant physiologic endpoint of cell death, in some cases the remnants left behind by death can also function to support tissue homeostasis. Here we discuss specific functions of the end products of cell death, and how "after-death" functions may contribute to the roles of programmed cell death in physiology.

Mechanisms and consequences of entosis.

Multiple mechanisms have emerged where the engulfment of whole live cells, leading to the formation of what are called 'cell-in-cell' structures, induces cell death. Entosis is one such mechanism that drives cell-in-cell formation during carcinogenesis and development. Curiously, entotic cells participate actively in their own engulfment, by invading into their hosts, and are then killed non-cell-autonomously. Here we review the mechanisms of entosis and entotic cell death and the consequences of entosis on cell populations.

IL-15 induces strong but short-lived tumor-infiltrating CD8 T cell responses through the regulation of Tim-3 in breast cancer.

IL-15 has pivotal roles in the control of CD8(+) memory T cells and has been investigated as a therapeutic option in cancer therapy. Although IL-15 and IL-2 share many functions together, including the stimulation of CD8 T cell proliferation and IFN-γ production, the different in vivo roles of IL-15 and IL-2 have been increasingly recognized. Here, we explored the different effects of IL-15 and IL-2 on tumor-infiltrating (TI) T cells from resected breast tumors. We found that neither IL-2 nor IL-15 induced intratumoral CD8 T cell proliferation by itself, but after CD3/CD28-stimulation, IL-15 induced significantly higher proliferation than IL-2 during early time points, at day 2, day 3 and day 6. However, the IL-15-induced proliferation leveled off at day 9 and day 12, whereas IL-2 induced lower but progressive proliferation at each time point. Furthermore, IL-15 caused an early and robust increase of IFN-γ in the supernatant of TI cell cultures, which diminished at later time points, while the IL-2-induced IFN-γ production remained constant over time. In addition, the IL-15-costimulated CD8 T cells presented higher frequencies of apoptotic cells. The diminishing IL-15-induced response was possibly due to regulatory and/or exhaustion mechanisms. We did not observe increased IL-10 or PD-1 upregulation, but we have found an increase of Tim-3 upregulation on IL-15-, but not IL-2-stimulated cells. Blocking Tim-3 function using anti-Tim-3 antibodies resulted in increased IL-15-induced proliferation and IFN-γ production for a prolonged period of time, whereas adding Tim-3 ligand galectin 9 led to reduced proliferation and IFN-γ production. Our results suggest that IL-15 in combination of Tim-3 blocking antibodies could potentially act as an IL-2 alternative in tumor CD8 T cell expansion in vitro, a crucial step in adoptive T cell therapy.

Ultrasmall integrin-targeted silica nanoparticles modulate signaling events and cellular processes in a concentration-dependent manner.

Cellular and molecular-level interactions of nanoparticles with biological systems are a rapidly evolving field requiring an improved understanding of endocytic trafficking as the principal driver and regulator of signaling events and cellular responses. An understanding of these processes is vital to nanomedicine applications. Studies investigating the complex interplay of these processes and their relationship to targeted nanoparticles exploiting endocytic pathways are notably lacking. It is known that integrins traffic through the endosomal pathway and participate in diverse roles controlling signal transduction, cell migration, and proliferation. Here, it is shown that ultrasmall, nontoxic, core-shell silica nanoparticles (C-dots), surface-functionalized with cRGDY peptides, modestly activate integrin-signaling pathways, in turn, promoting the enhancement of cellular functions. First, nanomolar concentrations, two orders of magnitude higher than clinical trial doses, internalize within αvß3 integrin-expressing melanoma and endothelial cells, predominantly through an integrin receptor-dependent endocytic route. Second, integrin-mediated activation of focal adhesion kinase (FAK) and downstream signaling pathways occurs, in turn, upregulating phosphorylated protein expression levels and promoting concentration-dependent cellular migration and proliferative activity. Inhibiting FAK catalytic activity leads to decreased phosphorylation levels and cellular migration rates. These findings may inform the design of more effectively-targeted nanomedicines and provide insights into endocytic regulation of signal transduction.

Autophagy proteins regulate cell engulfment mechanisms that participate in cancer.

Recent evidence has uncovered cross-regulation of mechanisms of cell engulfment by proteins of the autophagy pathway, in what is called LC3-Associated Phagocytosis, or LAP. By LAP, lysosome fusion to phagosomes and the degradation of engulfed extracellular cargo are facilitated by autophagy proteins that lipidate LC3 onto phagosome membranes. Here we discuss the contexts where LAP is known to occur by focusing on potential roles in tumorigenesis, including predicted consequences of LAP inhibition.

Entosis: the core mechanism and crosstalk with other cell death programs.

Cell death pathways play critical roles in organism development and homeostasis as well as in the pathogenesis of various diseases. While studies over the last decade have elucidated numerous different forms of cell death that can eliminate cells in various contexts, how certain mechanisms impact physiology is still not well understood. Moreover, recent studies have shown that multiple forms cell death can occur in a cell population, with different forms of death eliminating individual cells. Here, we aim to describe the known molecular mechanisms of entosis, a non-apoptotic cell engulfment process, and discuss signaling mechanisms that control its induction as well as its possible crosstalk with other cell death mechanisms.

Ferroptosis-like cell death promotes and prolongs inflammation in Drosophila.

Ferroptosis is a distinct form of necrotic cell death caused by overwhelming lipid peroxidation, and emerging evidence indicates a major contribution to organ damage in multiple pathologies. However, ferroptosis has not yet been visualized in vivo due to a lack of specific probes, which has severely limited the study of how the immune system interacts with ferroptotic cells and how this process contributes to inflammation. Consequently, whether ferroptosis has a physiological role has remained a key outstanding question. Here we identify a distinct, ferroptotic-like, necrotic cell death occurring in vivo during wounding of the Drosophila embryo using live imaging. We further demonstrate that macrophages rapidly engage these necrotic cells within the embryo but struggle to engulf them, leading to prolonged, frustrated phagocytosis and frequent corpse disintegration. Conversely, suppression of the ferroptotic programme during wounding delays macrophage recruitment to the injury site, pointing to conflicting roles for ferroptosis during inflammation in vivo.

Stress-induced microautophagy is coordinated with lysosome biogenesis and regulated by PIKfyve.

Lysosome turnover and biogenesis are induced in response to treatment of cells with agents that cause membrane rupture, but whether other stress conditions engage similar homeostatic mechanisms is not well understood. Recently we described a form of selective turnover of lysosomes that is induced by metabolic stress or by treatment of cells with ionophores or lysosomotropic agents, involving the formation of intraluminal vesicles within intact organelles through microautophagy. Selective turnover involves noncanonical autophagy and the lipidation of LC3 onto lysosomal membranes, as well as the autophagy gene-dependent formation of intraluminal vesicles. Here, we find a form of microautophagy induction that requires activity of the lipid kinase PIKfyve and is associated with the nuclear translocation of TFEB, a known mediator of lysosome biogenesis. We show that LC3 undergoes turnover during this process, and that PIKfyve is required for the formation of intraluminal vesicles and LC3 turnover, but not for LC3 lipidation onto lysosomal membranes, demonstrating that microautophagy is regulated by PIKfyve downstream of noncanonical autophagy. We further show that TFEB activation requires noncanonical autophagy but not PIKfyve, distinguishing the regulation of biogenesis from microautophagy occurring in response to agents that induce lysosomal stress.

Engineering focusing on cancer.

While cancer research and care have benefited from revolutionary advances in the ability to manipulate and study living systems, the field is limited by a lack of synergy to leverage the power of engineering approaches. Cancer engineering is an emerging subfield of biomedical engineering that unifies engineering and cancer biology to better understand, diagnose, and treat cancer. We highlight cancer engineering's unique challenges, the importance of creating dedicated centers and departments that enable translational collaboration, and educational approaches to arm a new generation of scientists with engineering expertise and a fundamental understanding of cancer biology to transform clinical cancer care.

The story behind the emergence of different forms of cell death.

Various types of cell death program are needed for cells to respond to changes in physiological conditions. In this collection of Voices, we asked scientists to tell the story behind their contributions to the identification and mechanistic dissection of cell death pathways and to discuss future directions for such research.

Tumorigenesis driven by the BRAFV600E oncoprotein requires secondary mutations that overcome its feedback inhibition of migration and invasion.

BRAFV600E mutation occurs in 46% of melanomas and drives high levels of ERK activity and ERK-dependent proliferation. However, BRAFV600E is insufficient to drive melanoma in GEMM models, and 82% of human benign nevi harbor BRAFV600E mutations. We show here that BRAFV600E inhibits mesenchymal migration by causing feedback inhibition of RAC1 activity. ERK pathway inhibition induces RAC1 activation and restores migration and invasion. In cells with BRAFV600E, mutant RAC1, overexpression of PREX1, PREX2, or PTEN inactivation restore RAC1 activity and cell motility. Together, these lesions occur in 48% of BRAFV600E melanomas. Thus, although BRAFV600E activation of ERK deregulates cell proliferation, it prevents full malignant transformation by causing feedback inhibition of cell migration. Secondary mutations are, therefore, required for tumorigenesis. One mechanism underlying tumor evolution may be the selection of lesions that rescue the deleterious effects of oncogenic drivers.

Dose-dependent induction of distinct phenotypic responses to Notch pathway activation in mammary epithelial cells.

Aberrant activation of Notch receptors has been implicated in breast cancer; however, the mechanisms contributing to Notch-dependent transformation remain elusive because Notch displays dichotomous functional activities, promoting both proliferation and growth arrest. We investigated the cellular basis for the heterogeneous responses to Notch pathway activation in 3D cultures of MCF-10A mammary epithelial cells. Expression of a constitutively active Notch-1 intracellular domain (NICD) was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. Interestingly, we found that these heterogeneous phenotypes reflect differences in Notch pathway activation levels; high Notch activity caused down-regulation of multiple matrix-adhesion genes and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. Moreover, microarray analyses implicated NICD-induced p63 down-regulation in loss of matrix adhesion. In addition, a reverse-phase protein array-based analysis and subsequent loss-of-function studies identified STAT3 as a dominant downstream mediator of the NICD-induced outgrowth. These results indicate that the phenotypic responses to Notch are determined by the dose of pathway activation; and this dose affects the balance between growth-stimulative and growth-suppressive effects. This unique feature of Notch signaling provides insights into mechanisms that contribute to the dichotomous effects of Notch during development and tumorigenesis.

MiR-130b modulates the invasive, migratory, and metastatic behavior of leiomyosarcoma.

Leiomyosarcoma (LMS) is an aggressive, often poorly differentiated cancer of the smooth muscle (SM) lineage for which the molecular drivers of transformation and progression are poorly understood. In microRNA (miRNA) profiling studies, miR-130b was previously found to be upregulated in LMS vs. normal SM, and down-regulated during the differentiation of mesenchymal stem cells (MSCs) into SM, suggesting a role in LMS tumor progression. In the present study, the effects of miR-130b on human LMS tumorigenesis were investigated. Stable miR-130b overexpression enhanced invasion of LMS cells in vitro, and led to the formation of undifferentiated, pleomorphic tumors in vivo, with increased growth and metastatic potential compared to control LMS cells. TSC1 was identified as a direct miR-130b target in luciferase-3'UTR assays, and shRNA-mediated knockdown of TSC1 replicated miR-130b effects. Loss-of-function and gain-of-function studies showed that miR-130b levels regulate cell morphology and motility. Following miR-130b suppression, LMS cells adopted a rounded morphology, amoeboid mode of cell movement and enhanced invasive capacity that was Rho/ROCK dependent. Conversely, miR-130b-overexpressing LMS cells exhibited Rho-independent invasion, accompanied by down-regulation of Rho-pathway effectors. In mesenchymal stem cells, both miR-130b overexpression and TSC1 silencing independently impaired SM differentiation in vitro. Together, the data reveal miR-130b as a pro-oncogenic miRNA in LMS and support a miR-130b-TSC1 regulatory network that enhances tumor progression via inhibition of SM differentiation.