Autophagy and Ferroptosis - What's the Connection?

Abstract

Purpose of Review

Autophagy is a conserved intracellular degradation system and plays a dual role in cell death, depending on context and phase. Ferroptosis is a new form of regulated cell death that mainly depends on iron accumulation and lipid peroxidation. In this review, we summarize the processes of autophagy and ferroptosis and discuss their crosstalk mechanisms at the molecular level.

Recent Findings

The original study shows that ferroptosis is morphologically, biochemically, and genetically distinct from autophagy and other types of cell death. However, recent studies demonstrate that activation of ferroptosis is indeed dependent on the induction of autophagy. Additionally, many ferroptosis regulators such as SLC7A11, GPX4, NRF2, p53, HSPB1, CISD1, FANCD2, and ACSL4 have been identified as potential regulators of autophagy.

Summary

This review not only highlights the importance of autophagy as an emerging mechanism of ferroptosis, but also raises new insights regarding regulated cell death.

Introduction

Different types of cell death are often defined by distinct morphological, biochemical, genetic, and functional mechanisms [1]. In recent years, the scientific world has discovered several new types of regulated cell death (RCD). These types of RCD are implicated in both physiological and pathological processes in human health and disease [2]. Research continues to focus on the molecular machinery and signaling pathways that control various types of RCD, as well as the crosstalk between different types of RCDs [3, 4]. The field of RCD research keeps advancing at such an alarming rate. At present, 11 different types of RCD have been identified: anoikis, autophagic cell death, apoptosis, cornification, entosis, ferroptosis, mitotic catastrophe, necroptosis, netosis, parthanatos, and pyroptosis [5]. Among them, ferroptosis, an iron-dependent form of RCD, is the latest to be identified in 2012 [6]. It has become increasingly evident that iron accumulation and subsequent lipid peroxidation play a critical role in mediating ferroptosis [7]. Thus, various molecules and signals involved in iron metabolism and lipid peroxidation contribute to ferroptosis regulation.

The original study shows that ferroptosis is distinct from apoptosis, necrosis, and autophagy in cancer cells [6]. However, recent studies demonstrate that activation of autophagy is required for the induction of ferroptosis not only in cancer cells, but also in normal cells [8–10]. Unraveling how autophagy regulates ferroptosis will not only reveal fundamental mechanistic insights into this type of RCD, but also provide new therapeutic targets for the treatment of ferroptosis-associated diseases. This review briefly introduces the processes of ferroptosis and autophagy and focuses on the emerging molecular interactions between these processes.

Autophagy: a lysosome-dependent degradation pathway

Autophagy (from the Greek for “self-eating”) is an evolutionarily-conserved homeostatic mechanism that includes three general subtypes: macroautophagy, microautophagy, and chaperone-mediated autophagy [11]. Macroautophagy (hereafter referred to as autophagy), as one of the lysosome-dependent degradation pathways, is involved in the removal of not only aging proteins, but also damaged organelles, as well as invading pathogens. To that end, autophagy is a critical regulator of cellular homeostasis and is involved in various pathologic conditions including infection, immunity, metabolism, and cancer [12]. The term “autophagy” was first introduced in the 1960s; however, the molecular mechanism of autophagy remained obscure until the 1990s. In 1993, Yoshinori Ohsumi, a Japanese cell biologist, identified the first autophagy-regulated (ATG) mutant that could not accumulate autophagic bodies in the vacuole and reported that 15 ATG genes are required for the activation of autophagy in eukaryotic cells [13]. He was therefore awarded the 2016 Nobel Prize in Physiology or Medicine for his pioneering studies on the molecular machinery of autophagy [14]. To date, 41 ATG genes have been identified as controlling autophagy in yeast; approximately half of these genes are clearly conserved in humans [15]. ATG proteins can form different complexes by posttranslational modifications to control the formation of three main subcellular structures of autophagy: the phagophore, the autophagosome, and the autolysosome [16, 17].

Autophagy plays a dual role either preventing or promoting death [18]. In many cases, autophagy-related stress tolerance can enable cell survival under various types of cellular death stimuli. In some cases, excessive or uncontrolled levels of autophagy can trigger autophagy-dependent cell death, termed “autophagic cell death” [19]. Distinguishing the various functions of autophagy in cell life and death remains challenging [20, 21].

Ferroptosis: an iron-dependent lipid peroxidation pathway

The molecular characterization of various cancers has shown that cancers with the same origins, pathologic stages, and clinical stages can be greatly heterogeneous in their patterns of genetic alterations, epigenetic changes, and gene expressions. One goal of precision medicine is to search for genotype-selective agents that become lethal to tumor cells only in the presence of specific genetic alterations [22]. Mutations in the small GTPase RAS of proto-oncogenes (e.g., K-RAS, H-RAS, and N-RAS) are very common in human cancer [23]. Ferroptosis was originally identified as a form of RAS mutation-dependent RCD by Brent Stockwell’s lab at Columbia University in 2012 [6, 24]. Ferroptosis can be induced by RAS-selective lethal (RSL) small molecular compounds such as erastin and RSL3 [25, 24]. Moreover, several anticancer drugs (e.g., sulfasalazine, sorafenib, and artesunate) also have the ability to induce ferroptosis [26–28]. In addition to cancer cells, ferroptosis can be observed in normal cells under some conditions [29–32]. Thus, ferroptosis is implicated in wide variety of physiological and pathological processes.

Stockwell’s group showed that ferroptosis is morphologically, biochemically, and genetically distinct from apoptosis, necroptosis, and autophagy. Transmission electron microscopy (TEM) analysis shows that ferroptotic cells undergo a different substructural change [6]. Onset of apoptosis (e.g., chromatin condensation), necrosis (e.g., rupture of the plasma membrane), and autophagy (e.g., double membrane structures) are not observed during ferroptosis by TEM assay [6]. In contrast, cells undergoing ferroptosis display characteristic changes in mitochondrial size (e.g., small size) and structure (e.g., reduced mitochondrial crista density and increased mitochondrial membrane potential) [6]. The activated caspases (the apoptosis effector) and receptor interacting serine/threonine kinase (“RIPK”, the necroptosis effector) are not observed in erastin-induced ferroptosis. ZVAD-FMK (an inhibitor of pan-caspase), necrostatin-1 (a selective inhibitor of RIPK1), and chloroquine (an inhibitor of autophagy by raising the lysosomal pH) cannot block ferroptosis [6]. In contrast, iron accumulation and lipid peroxidation are critical signaling events that drive ferroptosis [33]. Intracellular iron chelate (e.g., deferoxamine and desferrioxamine mesylate) and various antioxidants (e.g., vitamin E, liproxstatin-1, ferrostatin-1, and baicalein) prevent ferroptosis [6, 34, 32, 35]. Several genes and proteins have recently been demonstrated to be involved in ferroptosis by modulating the generation of reactive oxygen species (ROS)-mediated lipid peroxidation by iron and lipid (Figure 1). However, these critical players in ferroptosis factors also exhibit the ability to regulate autophagy and autophagic cell death (see below).

Figure 1.

Molecular regulators of ferroptosis.

Autophagy contributes to ferroptosis by ferritinophagy

We and other groups recently provided confidence data that ferroptosis is an autophagic cell death process [9, 8, 10]. Mechanistically, selective autophagy (namely ferritinophagy) contributes to ferroptosis by mediating the degradation of ferritin. Ferritin is composed of ferritin heavy chain (FTH) and ferritin light chain (FTL). They regulate iron metabolism by sequestering and storing iron in non-toxic and bioavailable forms, which prevents harmful oxidative reactions. FTH catalyzes the first step in iron storage, the oxidation of Fe²⁺, whereas FTL promotes the nucleation of ferrihydrite, enabling storage of Fe³⁺. The expression of ferritin is downregulated in ferroptosis-sensitive cells compared to ferroptosis-resistant cells, suggesting that ferritin negatively regulates ferroptosis [25].

The clearance of aggregated proteins by autophagy requires various cargo receptor proteins linking cargo to growing autophagosomal membranes. Quantitative proteomics identifies nuclear receptor coactivator 4 (NCOA4) as the cargo receptor responsible for ferritin degradation by ferritinophagy [36]. Degradation of ferritin within autolysosomes ultimately results in the release of chelated iron, which is subsequently transported back into the cytosol to induce oxidative stress. Like knockout or knockdown of ATGs (e.g., ATG5, and ATG7), genetic depletion of NCOA4 by specific shRNA limits ferritin degradation and subsequent ferroptosis in normal fibroblasts and tumor cells [8], whereas forced expression of NCOA4 by cDNA transfection accelerates ferroptosis by induction of ferritin degradation [8]. These findings indicate that selective autophagy plays a specific role in mediating ferroptosis. In addition to iron buffering, FTH also enhances thymidine biosynthesis [37]. It is unclear whether thymidine biosynthesis is regulated by ferritinophagy, which affects the process of ferroptosis.

Molecular interactions between ferroptosis and autophagy

SlC7A11

System Xc⁻, an amino acid antiporter on the cellular surface, is involved in antioxidant defense through regulating glutamate, cysteine, and glutathione (GSH) metabolism. As a functional core component of system Xc⁻, solute carrier family 7 member 11 (SLC7A11) plays an important role in protection against ferroptosis by inhibition of lipid peroxidation [6]. In contrast, pharmacologic inhibition of SCL7A11 by erastin, sulfasalazine, and sorafenib significantly cause intracellular GSH depletion, which contribute to lipid peroxidation and subsequent ferroptosis [6]. Research studies have shown that GSH also can inhibit basal and induced autophagy by starvation or oxidative stress, suggesting a potential role of GSH in the modulation of crosstalk between ferroptosis and autophagy [38, 39].

GPX4

Glutathione peroxidase (GPX) is an antioxidant enzyme family that includes GPX1-8 in humans. They can scavenge hydrogen and lipid peroxides under oxidative stress. Compared with other GPX members, GPX4 plays a unique role in the inhibition of ferroptosis in vitro and in vivo. Loss of GPX4 expression or activity by genetic or drug approaches promotes ferroptosis through a lipid ROS-dependent manner [35]. In contrast, upregulation of GPX4 expression can diminish lipid ROS-induced ferroptosis [35]. In mice, GPX4 conditional knockout in kidney, T cells, or brain exhibits tissue injury and immune dysfunction associated with increased ferroptosis [32, 30, 40]. In contrast, ferroptosis inhibitors such as liproxstatin-1 can reverse these abnormalities including oxidative injuries in GPX4 conditional knockout mice [32, 30], supporting that excessive ferroptosis play a pathologic role in tissue injury, inflammation and immune response in vivo. However, GPX4 may have roles as modulators of non-ferroptotic RCD such as apoptosis, necroptosis, and autophagy. Consistent with their enhanced antioxidant ability, GPX4 overexpression has been shown to inhibit ROS-mediated autophagy as well as immunogical cell death [41]. In addition, it remains of great interest to determine whether autophagy contributes to GPX4 protein degradation in ferroptosis [42, 43].

p53

p53, the most commonly mutated tumor-suppressor gene in human cancers, plays a critical role in the regulation of many aspects of cancer biology, including cell death. p53 regulates apoptosis and autophagy in both a transcription-dependent and -independent manner. For example, p53 in the cytosol blocks autophagy in a transcription-independent manner, whereas p53 in the nucleus activates autophagy in a transcription-dependent way [44]. These findings clearly demonstrate that the subcellular localization of p53 affects its activity in autophagy. Interestingly, recent studies indicate that nuclear p53 is required for ferroptosis in a transcription-dependent way. Nuclear p53 suppresses the expression of SLC7A11 and therefore increases lipid peroxidation [45]. Moreover, nuclear p53 can enhance spermidine/spermine N1-acetyltransferase 1 (SAT1) and glutaminase 2 (GLS2) expression in cancer cells, which results in ferroptosis by triggering lipid peroxidation [46]. This process of p53-mediated ferroptosis is highly regulated by p53 acetylation [47]. A better understanding of the transcription-dependent and -independent roles of p53 should give us key insight into the function of p53 in ferroptosis and tumor biology. The role of p53-mediated autophagy in p53-mediated ferroptosis remains unclear.

NRF2

The nuclear factor erythroid 2-related factor 2 (NRF2) is required for the activation of the cellular antioxidant response to oxidative or electrophilic stresses. This process is involved in increased NRF2 stabilization and subsequent NRF2-mediated gene transcription. Under normal conditions, NRF2 is mainly degraded by the kelch-like ECH-associated protein 1 (KEAP1)-mediated ubiquitin-proteasome pathway. Under oxidative or electrophilic stress, KEAP1-mediated degradation of NRF2 is inhibited; this process is also regulated by autophagy. More importantly, the cargo receptor p62 in autophagy activates NRF2 through inactivation of KEAP1 [48]. Our recent study also demonstrated that the binding of p62 to KEAP1 promotes NRF2 protein stability in hepatocellular carcinoma cells under sorafenib-induced ferroptosis [49]. As a result, activation of the p62-KEAP1-NRF2 pathway limits sorafenib-induced ferroptotic cell death. In particular, metallothionein-1G (a cysteine residues-rich protein) seems to be a key NRF2 target gene contributing to ferroptosis resistance in response to sorafenib in hepatocellular carcinoma cells [50]. These findings provide a potential molecular link between autophagy and ferroptosis in hepatocellular carcinoma cells.

HSPB1

Heat shock protein family B (small) member 1 (HSPB1, also termed HSP25 in mice or HSP27 in humans) is a member of the HSPs that is constitutively expressed in multiple cells or tissues. Under various types of environmental stress, the protein expression of HSPB1 is upregulated and confers cellular resistance to cell death types such as apoptosis. Moreover, phosphorylation of HSPB1 leads to a structural change of HSPB1 from a multimer to a dimer/monomer. This protein post-translational modification increases the ability of HSPB1-mediated actin polymerization and reorganization. Phosphorylated HSPB1 plays a different role in autophagy and apoptosis. Several studies have shown that phosphorylated HSPB1 is required for selective autophagy (e.g., mitophagy and lipophagy) [51–53]. However, phosphorylated HSPB1 induced by erastin blocks cytoskeleton-mediated iron uptake and subsequent lipid peroxidation under ferroptosis [54]. These functional differences may result from the different upstream kinase responses for the phosphorylation of HSPB1 at different sites.

CISD1

CDGSH iron sulfur domain 1 (CISD1, also called mitoNEET) is a mitochondrial protein located in the outer membrane. CISD1 plays an important role in mediating the crosstalk between mitochondrial iron uptake and oxidative stress in both normal and cancer cells. The human CISD1 is a novel target of pioglitazone, a type II diabetes drug. The upregulation of protein expression of CISD1 in cancer cells limits autophagic activity. In contrast, genetic or pharmacological inhibition of CISD1 remarkably increases autophagy in cancer cells [55, 56]. In addition to autophagy, upregulation of CISD1 also limits ferroptotic activity in cancer cells [57]. Knockdown of CISD1 by shRNA increased erastin-induced intramitochondrial lipid peroxidation and subsequent ferroptosis [57]. In contrast, the binding of pioglitazone to CISD1 stabilizes the Fe-S cluster, which blocks mitochondrial iron-mediated lipid peroxidation as well as ferroptosis [57]. These studies suggest that mitochondrial iron uptake through CISD1 is implicated in both autophagy and ferroptosis in cancer cells.

FANCD2

Fanconi anemia (FA) is a genetically heterogeneous recessive disorder due to defective DNA repair. Disruption of iron metabolism plays a key role in the etiology of Fanconi anemia. FANCD2, the central protein of the FA pathway, is activated by mono-ubiquitination in the DNA damage response. Loss of FANCD2 increases DNA damage and cancer incidence. Previous studies show that FANCD2-deficient cells are hypersensitive to oxidative stress and DNA crosses links due to impaired autophagy [58]. Despite that FANCD2-deficient bone marrow stromal cells (BMSCs) are hypersensitive to erastin-induced ferroptosis, the autophagic activity is not significantly changed by FANCD2 deficiency. These findings suggest an autophagy-independent role of FANCD2 in the regulation of ferroptosis in BMSCs. FANCD2-deficient BMSCs exhibit iron overload and lipid peroxidation in response to erastin [59]. In particular, loss of FANCD2 is associated with increased gene expression for iron uptake (e.g., transferrin, transferrin receptor, and HSPB1) and decreased gene expression for iron storage (e.g., FTH) and iron export (e.g., hepcidin antimicrobial peptide) in ferroptosis [59]. These data indicate a critical role of FANCD2 in the protection against ferroptosis in BMSCs.

ACSL4

Lipid peroxidation can be described generally as a core event of ferroptosis. However, the source and identity of lipid death signals that cause ferroptosis are poorly defined. We and other groups recently identified that the acyl-CoA synthetase long-chain family member 4 (ACSL4) plays a key role in the execution of ferroptosis [60–62]. ACSL4 (but not other ACSLs including ACSL1, ACSL3, ACSL5, and ACSL6) expression correlates with cellular sensitivity to erastin-induced ferroptosis [60]. Mechanically, ACSL4-mediated production of 5-hydroxyeicosatetraenoic acid and subsequent production of oxidized species of phosphatidylethanolamines is essential for ferroptosis [60–62]. These findings indicate that ACSL4 is a not only a biomarker, but also a contributor of ferroptosis [60]. Interestingly, ACSL4 is also involved in the regulation of the activity of mechanistic target of rapamycin (mTOR) complex I (mTORC1) and mTOR complex II (mTORC2) [63]. These two mTOR complexes have different functions in cell growth, metabolism, and autophagy. mTORC2 can inhibit autophagy by activation of mTORC1 [64]. The direct contribution of mTOR in ACSL4-mediated ferroptosis needs further investigation.

HSPA5

The heat shock 70kDa protein 5 (HSPA5, also termed GRP78 or BIP) is a member of the molecular chaperones expressed primarily in the endoplasmic reticulum. As a key component of the unfolded protein response, HSPA5 promotes cell survival under conditions of endoplasmic reticulum stress-induced autophagy. A recent study demonstrated that upregulation of HSPA5 is a negative regulator of ferroptosis in pancreatic cancer cells [65]. Increased HSPA5 expression limits lipid peroxidation in ferroptosis by directly protecting against GPX4 degradation [65].

Conclusion

In the context of biological diversity, multiple types of cell death exist and have been reported. As a novel RCD, ferroptosis occurs in cells when iron accumulation and lipid peroxidation is activated. A high level of ferroptosis not only selectively kills cancer cells, but also causes tissue injury. Autophagy as a degradation pathway plays a dual role - either pro-survival or pro-death - depending on many conditions. Activation of autophagy seems to contribute to ferroptosis. However, the mechanism of autophagy-mediated ferroptosis remains largely unknown. Another important area is delineating whether and how lipid peroxidation impacts autophagosome formation as a feedback loop.

List of Abbreviations

RCD

regulated cell death

ATG

autophagy-regulated

TEM

transmission electron microscopy

RSL

RAS-selective lethal

RIPK

receptor interacting serine/threonine kinase

ROS

reactive oxygen species

NCOA4

nuclear receptor coactivator 4

GSH

glutathione

SLC7A11

solute carrier family 7 member 11

GPX

glutathione peroxidase

SAT1

spermidine/spermine N1-acetyltransferase 1

GLS2

glutaminase 2

NRF2

nuclear factor erythroid 2-related factor 2

KEAP1

kelch-like ECH-associated protein 1

HSPB1

heat shock protein family B (small) member 1

CISD1

CDGSH iron sulfur domain 1

FA

fanconi anemia

ACSL4

acyl-CoA synthetase long-chain family member 4

mTOR

mechanistic target of rapamycin

FTH

ferritin heavy chain

FTL

ferritin light chain

BMSCs

bone marrow stromal cells

HSPA5

the heat shock 70kDa protein 5