Autophagy plays a multifaceted role in regulating both
Autophagy plays a multifaceted role in regulating both the quality and quantity of protein (e.g., protein half-life and activity) and organelles (e.g., mitochondrial number and function), thus determining cell fate . The induction of autophagy has been generally considered a programmed cell survival mechanism in response to various types of stress [16,17]. However, an uncontrolled or inappropriate autophagic response can also be damaging or lethal . Historically, the term autophagic cell death was used to describe type II cell death based on the morphological appearance of autophagic vesicles during cell death . Currently, the Nomenclature Committee on Cell Death defines autophagy-dependent cell death (ADCD) as a type of regulated cell death (RCD) that is precipitated or executed by the autophagy machinery . For example, the overexpression of Atg1 results in developmental cell death in the salivary gland in Drosophila . In addition to development, ADCD has been connected to multiple human diseases such as cancer, neurodegenerative diseases, and tissue injury [, , ]. The induction of ADCD by anticancer drugs such as BH3 mimetics (ABT737, obatoclax, gossypol, and Z36), histone deacetylase inhibitors (butyrate and suberoylanilide hydroxamic acid), or natural products (resveratrol and betulinic acid) can restore cell death in apoptosis-resistant SNX-2112 [, , , , , ].
Although its exact mechanism is not completely understood, ADCD activates alternative cell death pathways at different levels. First, excessive self-consumption of cytoplasmic components by bulk autophagy can result in overloading of lysosomes, with detrimental effects on the cell . Second, excessive removal of mitochondria by mitophagy may result in deficient energy production . Third, the induction of autosis by the activation of the Na+/K+-ATPase pump can cause a lethal electrolyte disturbance secondary to excessive ATP depletion . Autosis is an autophagy-dependent form of cell death triggered by autophagy-inducing peptides (e.g., Tat-Beclin 1), starvation, and neonatal cerebral hypoxia-ischemia . Fourth, the degradation of negative regulators of cell death by selective autophagy can trigger various types of RCD such as apoptosis , necroptosis  or ferroptosis .
Ferroptosis is a recently defined form of RCD driven by iron-dependent lipid peroxidation (Fig. 2), an area of investigation that has seen an enormous boost after the pioneering work of Stockwell and colleagues. The deregulation of ferroptosis triggers the development of numerous human diseases [37,38]. Although an early study reported that ferroptosis is distinct from other types of RCD, including ADCD at the biochemical, morphological, and genetic levels , accumulating evidence indicates that ferroptosis requires the autophagy machinery for its execution . In this review, we discuss recent progress with respect to the molecular mechanism of ferroptosis and its relationship with autophagy.
The discovery of ferroptosis Ferroptosis was initially discovered when screening small molecule compounds for targeting oncogenic RAS mutations. The three RAS oncogenes, including KRAS, NRAS, and HRAS, are the most frequently mutated oncogenes in human cancer. In 2003, Dolma et al. used engineered human BJ fibroblasts (BJ-TERT/LT/ST/RASV12 cells) that expressed various oncogenes, including the human catalytic subunit of the enzyme telomerase (TERT), a genomic construct encoding the simian virus 40 large (LT) and small T (ST) oncoproteins, and an oncogenic allele of HRAS (RASG12V) to screen 23,550 compounds to identify genotype-selective antitumor agents . This screen led to the identification of a small molecule compound termed ‘erastin’ that selectively induces nonapoptotic cell death in both an ST- and RASG12V-dependent manner . In 2007, Yagoda et al. further tested the genotype-selective antitumor activity of erastin in various RAS mutation cancer cell lines (e.g., BJ-TERT/LT/ST/RASV12, HT1080, and Calu-1 cells) and confirmed that the RAS-BRAF (B-Raf proto-oncogene, serine/threonine kinase)-MAP2K/MEK (mitogen-activated protein kinase kinase)-MAPK/ERK (mitogen-activated protein kinase) pathway that mediates oxidative stress, as well as VDAC (voltage-dependent anion channel) that mediates mitochondria dysfunction, are required for erastin-induced cell killing . This study also identified that VDAC2 and VDAC3 are direct targets of erastin  (Fig. 3). In 2012, Dixon et al. finally defined erastin-induced cell death as an iron-dependent RCD that was nicknamed ‘ferroptosis’ . Erastin-induced iron accumulation promotes reactive oxygen species (ROS) production, which results in lipid peroxidation and subsequent death . Erastin-induced death can be avoided by iron chelation or antioxidants, but not by inhibitors of caspase, cathepsin or calpain proteases, RIPK1 (receptor-interacting serine/threonine kinase 1), PPID/cyclophilin D, lysosomal function or autophagy . The genetic inhibition of cellular iron uptake, but not that of apoptosis effectors (such as BAX [BCL2-associated X, apoptosis regulator] and BAK1/BAK [BCL2 antagonist/killer 1]), also blocks erastin-induced cell death . These seminal observations from the Stockwell Laboratory established that ferroptosis is different from apoptosis, necroptosis and autophagy . However, pharmacologic and genetic insights from independent studies suggest that ferroptosis is a type of ADCD in some cancer cells (as we will discuss later). The reasons for the latter conclusion are unclear but might be due to tumor heterogeneity, drug stability, and the feedback loop. Further studies documented a crosstalk between ferroptosis and other established cell death modalities including apoptosis and necroptosis. For example, fibroblasts that are MLKL (mixed lineage kinase domain like pseudokinase)-deficient and necroptosis-resistant are more sensitive to erastin-induced ferroptosis . Erastin also induces apoptosis in lung (e.g., A549) and colorectal cancer cell lines (e.g., HT-29, DLD-1, and Caco-2) through the activation of TP53 (tumor protein p53) and mitochondrial oxidative injury [44,45] (Fig.3). Additionally, erastin promotes proliferation and differentiation of human peripheral blood mononuclear cells to B cells and NK cells through suppression of BMP (bone morphogenetic protein) family (BMP2, BMP4, BMP6, and BMP7) expression  (Fig.3). More recently, the major pro-ferroptosis activity of erastin has been linked to direct blocking system xc− (Fig.3) (as we will discuss later). These findings including raise questions about the theoretical underpinnings of ferroptosis.