Ferroptosis in Hepatocellular Carcinoma: From Bench to Bedside

Abstract

The most widespread type of liver cancer, hepatocellular carcinoma (HCC), is associated with disabled cellular death pathways. Despite therapeutic advancements, resistance to current systemic treatments (including sorafenib) compromises the prognosis of HCC patients, driving the search for agents that might target novel cell death pathways. Ferroptosis, a form of iron-mediated non-apoptotic cell death, has gained considerable attention as a potential target for cancer therapy, especially in HCC. The role of ferroptosis in HCC is complex and diverse. On one hand, ferroptosis can both contribute to the progression of HCC through its involvement in acute and chronic liver conditions. On the other hand, having ferroptosis affect HCC cells might be desirable. This review examines the role of ferroptosis in HCC from cellular, animal, and human perspectives, while examining its mechanisms, regulation, biomarkers, and clinical implications.

Introduction

The most prevalent primary liver cancer in adults is hepatocellular carcinoma (HCC), a global health concern with a rising incidence and mortality rate (1). HCC is more prevalent in sub-Saharan Africa and Southeast Asia compared to Western countries, frequently resulting from chronic liver disease caused by hepatitis B/C infection, alcohol abuse, or metabolic syndrome (e.g., non-alcoholic fatty liver disease [NAFLD]) (2). Despite the increasing utilization of surgical and locoregional treatments, approximately 50%-60% of HCC patients will undergo systemic therapies. Targeted therapies have constituted the main treatment approach for advanced-stage HCC for over a decade. The oral multi-tyrosine kinase inhibitors sorafenib and lenvatinib (and donafenib in China) confer median overall survival benefits of 11-14 months, and regorafenib, cabozantinib (which inhibit vascular endothelial growth factor [VEGF] receptor 2 [VEGFR2] and other tyrosine kinases), and ramucirumab (a monoclonal antibody [mAb] specific for VEGFR2) yield benefits in the order of 8-11 months overall survival in second-line treatment (3). The new standard of care for HCC is the combination of the immune checkpoint inhibitor atezolizumab (an anti-CD274/PD-L1 antibody) and VEGF blockade with bevacizumab (4). However, the development of drug resistance, which at least is partly due to disabled apoptosis, remains a significant challenge in clinical oncology. The deconvolution of such resistance mechanisms as well as the development of drugs that induce non-apoptotic cell death could provide new opportunities for HCC therapy.

Cell death is a crucial process that plays both physiological and pathological roles in human health and disease, including cancer. Historically, cell death was categorized based on morphological criteria into apoptosis (characterized by cell shrinkage and fragmentation into membrane-enclosed “apoptotic bodies”), necrosis (characterized by the swelling of organelles, plasma membrane rupture, and cell lysis), and autophagy (characterized by the formation of autophagic vacuoles containing organelles). Currently, cell death is classified into accidental cell death (ACD) and regulated cell death (RCD) (5). In contrast to ACD, RCD is characterized by well-defined molecular and cellular mechanisms that can be manipulated through pharmacological or genetic means (6). Ferroptosis was initially identified as a type of iron-dependent, non-apoptotic RCD in RAS-mutant engineered cells that can be selectively induced by the chemical compound erastin (7). Further research has revealed that the core mechanism of ferroptosis involves lipid peroxidation caused by oxidative damage, which can be avoided by a complex antioxidant system (8). The study of ferroptosis and its implications for human diseases, including liver disease (9) and HCC (10), represent a cutting-edge area in biomedicine (11, 12).

Here, we will summarize the mechanisms of, and defenses against, ferroptosis, outline the regulators of ferroptosis, discuss biomarkers that may predict ferroptosis sensitivity in HCC, and analyze the potential use of ferroptosis-based scoring systems in clinical HCC practice. Finally, we will outline current strategies to pharmacologically target ferroptosis.

The activation mechanism of ferroptosis

Ferroptosis is a type of RCD caused by unrestricted lipid peroxidation and resultant membrane damage, dependent on iron accumulation (13). Ferroptosis differs in morphological, biochemical, and genetic aspects from other RCDs such as apoptosis, necroptosis, and pyroptosis (6). Ferroptosis can be triggered in different fashions, namely, (i) through the extrinsic pathway (e.g., the inhibition of amino acid antiporter system xc⁻ or the activation of iron transporter transferrin [TF] and lactotransferrin on cell membrane) and (ii) the intrinsic pathway (mainly by blocking the expression or activity of intracellular glutathione peroxidase 4 [GPX4]) (14). Ferroptosis does not require the main executors of apoptosis (driven by a specific subset of caspases), necroptosis (mediated by mixed-lineage kinase domain-like pseudokinase [MLKL]), and pyroptosis (executed by gasdermin D [GSDMD]) (7). Upon modification or cleavage, MLKL or GSDMD assemble into multimeric structures that function as porin proteins, facilitating the permeabilization and damage of cellular membranes. Rather, ferroptosis is driven by lipid peroxidation products, among which 4-hydroxy 2-nonenal (4HNE) is particularly toxic and abundant (15). This highlights that ferroptosis does not rely on known pore protein for lethal plasma membrane rupture to occur. In the forthcoming paragraphs, we will summarize the three key steps that ignite ferroptosis in cancer, including in HCC (Fig. 1).

Figure 1. The core mechanism and regulation of ferroptosis in HCC.

Ferroptosis is a type of cell death triggered by lipid peroxidation, a process that causes damage to cellular membranes. This process occurs in three stages: the generation of reactive oxygen species (ROS), the accumulation of polyunsaturated fatty acids (PUFAs), and the activation of enzymes involved in lipid peroxidation. There are multiple sources of these molecules. To counter ferroptosis, cells employ different defense systems. For instance, the production of monounsaturated fatty acids (MUFAs) through the SREBP1-SCD pathway can effectively inhibit PUFAs and their derivatives produced by the ACSL4-LPCAT3, ACSL4-SOAT1, and SLC1A5-GLS2 metabolic pathways. Additionally, antioxidant pathways in the cytoplasm, plasma membrane, and mitochondria work to prevent lipid peroxidation in a context-dependent manner. Finally, any damage to the membrane can be repaired through the action of the flippase SLC47A1 or the ESCRT-III machinery.

Generation of free radicals

Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species, are unstable molecules with an unpaired electron that can cause oxidative damage to cells, tissues, and DNA. ROS, which can be generated as a result of environmental factors (e.g., radiation, pollution, and tobacco smoke) and normal metabolic processes, favors the ignition of ferroptosis. The main ferroptosis-relevant sources of ROS are mitochondria, enzymes of the NADPH oxidase (NOX) family, and the iron-mediated Fenton reaction. Mitochondria are more abundant in hepatocytes than in any other cell type. Mitochondria generate the majority of cellular ROS as a side product of oxidative phosphorylation (OXPHOS). In the liver, NOX enzymes are expressed by immune cells endowed with the capacity of phagocytosis, as well as by non-phagocytic cells (16). NOX enzymes generate superoxide and other downstream forms of ROS in the plasma membrane. Finally, an excess of free reactive iron (II; Fe²⁺) can damage cells via the Fenton reaction with hydrogen peroxide (H₂O₂), yielding hydroxide (OH⁻) and hydroxyl (OH^•) radicals.

The level of free iron in cells is regulated by an integrated system controlling iron absorption, storage, utilization, and outflow. In the liver, iron-induced oxidative stress can lead to fibrosis and cirrhosis, which increases the risk of HCC (17). Ferroptosis can be caused by an increase in TF-mediated iron absorption or by autophagy-dependent degradation of ferritin, which is a protein that stores and inactivates iron (18, 19). However, TF present in hepatocytes may attenuate high iron diet-induced liver fibrosis and cirrhosis (20), suggesting that it may play an organ-specific hepatoprotective role.

The release of Fe²⁺ into the extracellular space requires iron transporters, such as solute carrier family 40 member 1 (SLC40A1, also known as ferroportin-1) and another iron transporter, lipocalin 2 (LCN2) in HCC cells. Fe²⁺ transported out of the cell by SLC40A1 need to be oxidized by ferroxidase (e.g., ceruloplasmin [CP]) to facilitate Fe³⁺ loading on TF. The deletion of CP can promote erastin- or RSL3-induced ferroptosis in HCC cells by preventing iron release (21). In contrast, the upregulation of LCN2 increases iron release, thereby preventing ferroptotic cell death in HCC cells (22). The expression of CDGSH iron sulfur domain 1 (CISD1), a mitochondrial iron-sulfur protein, negatively impacts the survival rate of patients with HCC (23) (Table 1). Additionally, CISD1 can prevent iron-triggered intra-mitochondrial lipid peroxidation and ferroptosis in HCC cells (24). These findings elucidate (part of) the link between disturbances in iron metabolism and susceptibility to ferroptosis in HCC.

Table 1.

Regulators of ferroptosis in HCC

Name	Function in ferroptosis	Expression in HCC patients	Function in liver tumorigenesis	References
ACSL4	Promoter; increases PUFA	Upregulation	Promoter	(26, 31, 32)
AIFM2	Suppressor; increases CoQ10 and membrane repair	Upregulation	Suppressor	(53, 56)
BECN1	Promoter; increases autophagy	Upregulation	Suppressor	(144, 145)
CISD1	Suppressor; inhibits mitochondrial iron	Upregulation	Promoter	(23, 24)
DHODH	Suppressor; increases CoQ10	Upregulation	Promoter	(57)
GLS2	Promoter; increases glutamate production	Downregulation	Suppressor	(87)
GPX4	Suppressor; inhibits lipid peroxidation	Upregulation	N/A	(48)
LCN2	Suppressor; promotes iron release	Upregulation	Promoter	(22, 146)
NCOA4	Promoter; increases iron	Upregulation	N/A	(147)
NFE2L2	Suppressor; inhibits oxidative damage	Upregulation	Suppressor or promoter	(67, 69, 71)
POR	Promoter; increases lipid peroxidation	Downregulation	Suppressor	(37, 38)
SCD	Suppressor; increases MUFA	Upregulation	Promoter	(33, 34)
SLC47A1	Suppressor; promotes membrane repair	Upregulation	N/A	(60)
SLC7A11	Suppressor; increases cystine uptake	Upregulation	Promoter	(47)
SREBP1	Suppressor; increase MUFA production	Upregulation	Promoter	(35, 148)
TF	Promoter; increases iron absorption	Upregulation	Suppressor	(20)
TP53	Suppressor or promoter; inhibits SLC7A11 or promotes PLTP/GLS2 expression	Upregulation or downregulation	Suppressor or promoter	(84, 85, 87, 90)
YAP1	Promoter; increases TFRC expression	Upregulation	Promoter	(96)
ZSCAN25	Suppressor; inhibits TP53 activity	Upregulation	Promoter	(86)

Poly- and monounsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are a type of lipid found in cell membranes that help maintain the membranes’ fluidity and stability by controlling the movement of membrane components. PUFAs also act as precursors to signaling molecules, such as eicosanoids, which regulate various physiological processes, including inflammation, coagulation, and blood pressure (25). The incorporation of PUFAs, such as arachidonic acid (AA) and adrenic acid (AdA), into phospholipids present in the cell membrane requires two key enzymes, acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) (26). ACSL4 converts AA/AdA into AA/AdA-CoA derivatives and promotes their esterification into phospholipids, while LPCAT3 catalyzes the conjugation of AA/AdA-CoA and membrane phosphatidylethanolamine (PE) to form AA/AdA–PE. A deficiency in the lipid flippase solute carrier family 47 member 1 (SLC47A1) promotes the use the ACSL4-sterol O-acyltransferase 1 (SOAT1) pathway instead of the ACSL4-LPCAT3 pathway to produce PUFA-containing cholesterol esters and promote ferroptosis (27). Other pathways, such as glutaminolysis-mediated α-ketoglutarate production (28), lipophagy-mediated degradation of lipid droplets (29), and peroxisome-driven biosynthesis of plasmalogens, (30) can provide an additional supply of PUFAs to enhance ferroptosis sensitivity in cancer cells, including in HCC. In contrast, tumor protein D52 (TPD52)-dependent lipid storage inhibits RSL3-induced ferroptosis in HCC cells (29). It is important to note that ACSL4 is expressed at elevated levels in the liver of NAFLD patients and likely plays a role in promoting the development of HCC (31, 32).

Monounsaturated fatty acids (MUFAs), such as oleic acid and palmitoleic acid, produced through the partial hydrogenation of unsaturated fatty acids during desaturation, can inhibit PUFA-related ferroptosis (33). MUFAs can also be obtained from dietary sources like olive oil, avocados, and nuts, or produced by the body through the action of stearoyl-CoA desaturase (SCD). SCD is overexpressed in surgically resected HCC tissues and acts as a major component in lipogenesis pathways (34). The inhibition of SCD reduces endogenous MUFA production and increases ferroptosis in Hep3B and Huh-7 cells (35). This process can be controlled by mitochondrial metabolism. Specifically, lactate uptake mediated by solute carrier family 16 member 1 (SLC16A1/MCT1, a monocarboxylate transporter) induces the upregulation of SCD in HCC cells, thereby inhibiting the activation of AMP-activated protein kinase (AMPK) and promoting subsequent expression of sterol regulatory element binding transcription factor 1 (SREBP1), a transcription factor for SCD mRNA expression (35). Of note, endogenously synthesized MUFAs probably contribute more to membrane lipids than MUFAs absorbed through nutrition. Thus, SCD is central for providing ferroptosis-inhibiting MUFAs. A major unresolved challenge is the lack of understanding regarding the intricate and fluctuating interactions between lipid metabolism and other metabolic processes in HCC.

Activation of lipid peroxidation

Lipid peroxidation can be initiated through both enzymatic and non-enzymatic mechanisms (25). Iron can increase the likelihood of ferroptosis by enhancing the activity of lipoxygenases (ALOXs), which are a class of non-heme iron-containing enzymes that catalyze the oxidation of PUFAs. This oxidation results in the formation of hydroperoxyl derivatives, such as lipid hydroperoxides (LOOHs) and reactive aldehydes, such as malondialdehyde and 4HNE. These aldehydes not only act as mediators of inflammation, but can also lead to the formation of adducts and crosslinks in proteins or DNA, eventually perturbing proteostasis and genomic stability (25). The mammalian ALOX family includes six members (ALOXE3, ALOX5, ALOX12, ALOX12B, ALOX15, and ALOX15B), each of which can play a context-dependent role in lipid peroxidation and ferroptosis (36).

Cytochrome P450 oxidoreductase (POR) is yet another enzyme that promotes PUFA peroxidation during ferroptosis (37). Unlike the ALOXs, which are expressed in a tissue-selective fashion, POR appears to be widely expressed in various tissues and has a broad-spectrum role in promoting lipid peroxidation. The POR-dependent lipid peroxidation process is initiated by the auto-oxidation of POR, which generates ROS in a reaction that is dependent on two co-factors: flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (37). These co-factors enhance the peroxidation of PUFAs during ferroptosis (37). Specifically, within HCC, the expression of POR is enhanced in tumor tissues, and this overexpression is associated with a poor prognosis (38). POR is also involved in regulating the expression of cytochrome P450 genes, as well as in controlling the localization and stability of cytochrome P450 enzymes within the cell (39). Future studies should investigate the impact of POR on the metabolism of ferroptosis-inducing drugs in the liver.

Of note, although lipid peroxidation is considered a key hallmark of ferroptosis, it is not an effector molecule of ferroptosis (40). Indeed, increases in oxidative stress-related iron and/or lipid metabolites are observed in many pathological conditions, which may not be related to ferroptosis (41). The point of no return for ferroptotic cells is unclear, one hypothesis may be irreversible damage to normal ion exchange pumps and membrane tension at the plasma membrane, leading to cellular edema and swelling (42, 43).

Defense mechanisms against ferroptosis

Defense against ferroptosis is mediated by antioxidant systems and membrane repair machinery that suppress lipid peroxidation and membrane rupture, respectively (Fig. 1).

The antioxidant system

The system xc⁻-glutathione (GSH)-GPX4 pathway plays a major role in antioxidant defense (44). Indeed, much of our understanding of the role and regulation of ferroptosis comes from the study of chemical inhibitors of system xc⁻ (e.g., erastin) or GPX4 (e.g., RSL3). System xc⁻, also known as the xCT antiporter, is a cell membrane transporter composed of solute carrier family 7 member 11 (SLC7A11) and solute carrier family 3 member 2 (SLC3A2). It facilitates the transport of cystine into the cell, which is then converted into cysteine and used in the synthesis of the antioxidant GSH (45). GSH is crucial for the activity of GPX4, a selenoenzyme that acts as a key repressor of ferroptosis by reducing toxic phospholipid hydroperoxides (PLOOHs) to non-toxic phospholipid alcohols (PLOHs). Through alternative splicing, the single GPX4 gene yields three GPX4 isoforms, among which the cytosolic and mitochondrial (but not the nuclear) isoforms have been documented to inhibit ferroptosis (44, 46). In humans, GPX4 and SLC7A11 are expressed at higher levels in HCC tumor tissue compared to normal tissues (47, 48). Mice with a liver-specific deficiency of Gpx4 died shortly after birth due to extensive hepatocyte degeneration caused by ferroptosis, emphasizing the importance of GPX4 in liver development (49). However, in other mouse tissues, Gpx4 deficiency does not necessarily cause ferroptosis. For example, Gpx4 deficiency in myeloid cells enhances pyroptosis (rather than ferroptosis) during bacterial infection (50). Gpx4 deficiency in mouse erythroid precursors leads to functional inactivation of caspase 8 (CASP8) by glutathionylation, resulting in necroptosis (51). Similarly, a low expression of GPX4 in cancer cells does not necessarily result in increased sensitivity to ferroptosis (52), suggesting that other antioxidant systems may protect against ferroptosis.

Two antioxidant systems have been found to play a role similar to that of GPX4 in protecting against ferroptosis. The first is the apoptosis-inducing factor mitochondria-associated 2 (AIFM2; also known as FSP1)-coenzyme Q (CoQ10) axis in the plasma membrane, which was discovered through CRISPR screening in Gpx4-deficient cells (53). CoQ10, also known as ubiquinone, is a coenzyme that helps protect cells from damage caused by free radicals. The role of CoQ10 in apoptosis and ferroptosis is determined by AIFM2. After AIFM2 transfers from mitochondria to the plasma membrane, it loses its pro-apoptotic activity and acquires an anti-ferroptotic effect due to its ability to reduce non-mitochondrial CoQ10 to ubiquinol using NADPH (53). AIFM2 can also reduce vitamin K to promote coagulation (54) or mediate cell membrane repair (55), suggesting that the inhibition of ferroptosis by AIFM2 may involve several mechanisms. Interestingly, increased expression of AIFM2 is positively associated with both overall survival and disease-specific survival in HCC (56), suggesting that AIFM2 may act as a tumor suppressor.

The second antioxidant system that backs up GPX4 in ferroptosis suppression is the dihydroorotate dehydrogenase (DHODH)-CoQ10 system in mitochondria, identified in cancer cells with low GPX4 expression (52). The enzyme DHODH converts CoQ10 to ubiquinol, which combats lipid peroxidation and protects cancer cells from ferroptosis (52). Among solid cancers, HCC has the highest levels of DHODH mRNA expression, and DHODH overexpression is correlated with tumor progression and metastasis (57). StAR-related lipid transfer domain containing 7 (STARD7), which is dual-localized to the intermembrane space of mitochondria and the cytosol after cleavage by the rhomboid protease presenilin-associated rhomboid-like (PARL), ensures the synthesis and transport of CoQ10 to the plasma membrane (58). Thus, STARD7 cleavage links the DHODH-CoQ10 system and the activation of the AIFM2-CoQ10 axis.

Recent advances in the study of ferroptosis have shown that stress proteins, such as nuclear protein 1 transcriptional regulator (NUPR1), can reduce the susceptibility to ferroptosis in HCC cells (59). Gaining a deeper understanding of the intricate relationships between various defense systems will shed light on the mechanisms regulating ferroptosis and could spur the development of targeted therapeutic strategies.

Membrane repair system

The membrane repair system is crucial for preserving the integrity and function of the plasma membrane, which is vital for cell survival and effective cellular communication. The system encompasses various pathways, such as those for the regrouping of lipids, the formation of lipid rafts, and the involvement of membrane-repair proteins like phospholipases, lipases, phosphatidylinositol transfer proteins, phosphatidylserine decarboxylases, and phospholipid flippases. SLC47A1, a lipid flippase, can prevent ferroptosis in cancer cells by facilitating lipid restructuring without affecting the transport of ferroptosis-inducing drugs such as erastin or RSL3 (27). An elevated expression of SLC47A1 has been observed in various cancers (including HCC), which may contribute to resistance to chemotherapy (60). As such, targeting SLC47A1 may be a promising approach to overcome resistance to ferroptosis in HCC cells.

The endosomal sorting complexes required for transport (ESCRTs) are multi-protein complexes involved in membrane trafficking and intracellular cargo sorting. They play a role in the formation and regulation of intraluminal vesicles within multivesicular endosomes, leading to the selective sorting and degradation of transmembrane proteins and lipids. The ESCRTs consist of four distinct complexes (ESCRT-0, -I, -II, and -III), each of which operates in a sequential and hierarchical manner to sort, package, and expel membrane cargos into intraluminal vesicles. The ESCRT-III machinery plays a crucial role in repairing the plasma membrane and preventing various forms of regulated necrosis, including necroptosis, pyroptosis, and ferroptosis (61, 62). The inhibition of charged multivesicular body protein 5 (CHMP5) or CHMP6, key components of ESCRT-III, increases ferroptosis susceptibility in HCC cells (63). The activation of ESCRT-III on cell membranes is dependent on calcium signaling, implying the role of calcium homeostasis and endoplasmic reticulum stress in the regulation of ferroptosis.

Ferroptosis inhibitors

Iron chelators, free radical-scavenging antioxidants, and ALOX inhibitors are the most commonly used inhibitors of ferroptosis (Fig. 1) (13). Two iron chelators are currently available and approved by the United States Food and Drug Administration: deferoxamine and deferasirox. These iron chelators effectively prevent ferroptosis by inhibiting iron-induced Fenton reaction and ROS generation. Ferrostatins (e.g., ferrostatin-1) and liproxstatins (e.g., liproxstatin-1) are radical-trapping antioxidants that slow down the rate of autoxidation of hydrocarbons (lipids) by reacting with chain-carrying peroxyl radicals (64). Most lipid peroxidation inhibitors, such as baicalein (65) and zileuton (66), target ALOX with variable activity in vitro and in vivo. Ferrostatins and liproxstatins may be more specific in preventing ferroptosis, and iron chelators and ALOX inhibitors have been previously reported to inhibit apoptosis and other cell deaths. Overall, the identification of ferroptosis requires the combination of different cell death inhibitors in vitro and in vivo.

Regulation of ferroptosis in HCC

In addition to the core regulators of ferroptosis summarized above, the susceptibility of HCC to ferroptosis can be regulated on a number of different levels, such as gene transcription, epigenetics, protein degradation, and solute macromolecule uptake.

Transcriptional mechanism

Ferroptosis involves the activation of multiple transcription factors. Below we summarize three relatively well-studied transcription factors involved in the regulation of ferroptosis in HCC (Fig. 2).

Figure 2. Master transcriptional regulation of ferroptosis in HCC.

(A) NFE2L2 is a crucial anti-ferroptotic transcription factor in HCC cells, as it regulates multiple genes involved in enhancing antioxidant capacity, reducing iron toxicity, and facilitating drug efflux. The expression of NFE2L2 is primarily controlled by the KEAP1-mediated degradation pathway through the proteasomal system. The level of KEAP1 is, in turn, regulated by autophagic degradation via SQSTM1. Multiple upstream kinases or enzymes regulate the sensitivity to ferroptosis by controlling the SQSTM1-KEAP1-NFE2L2 pathway in HCC cells. (B) TP53 plays a dual role in ferroptosis, depending on the expression of its target genes. Under normal circumstances, TP53 is degraded by MDM2. If MDM2 is absent, the stability of TP53 increases and its transcriptional function is further amplified through Ser 46 phosphorylation and the presence of the P47S variant. In addition to boosting PLTP expression, TP53-mediated inhibition of DPP4 leads to the suppression of ferroptosis. (C) YAP1 promotes ferroptosis by inducing the expression of TFRC, which increases iron uptake. The O-GlcNAcylation of YAP1, which is mediated by O-GlcNAc, enhances the activity of YAP1 and its ability to induce TFRC expression in HCC cells.

NFE2L2

The transcription factor NFE2-like BZIP transcription factor 2 (NFE2L2), also known as NRF2, plays a key role in maintaining redox balance and controlling antioxidant genes by binding to antioxidant response elements (AREs). The expression of NFE2L2 is elevated in HCC and has been implicated in promoting cell proliferation and advancing tumor progression, making it an independent predictor of outcomes in HCC patients (67). Furthermore, mutations in NFE2L2 (occurring in 5.1% of patients with HCC) have been implicated in HCC pathology (68). However, in Nfe2l2-deficient mice, chronic exposure to non-genotoxic hepatocarcinogens, such as pentachlorophenol and piperonyl butoxide, triggers oxidative stress and stimulates the development of pre-neoplastic lesions as well as their progression to HCC (69). These findings support the notion that oxidative stress has a dual role in liver tumorigenesis.

The protein NFE2L2 is involved in conferring cellular resistance against ferroptosis therapy in HCC (Fig. 2A). Ferroptosis activators, such as erastin and sorafenib, can increase NFE2L2 protein stability in HCC cells. This process is regulated by the autophagy receptor sequestosome 1 (SQSTM1/p62) that increases NFE2L2 expression through the inactivation of kelch-like ECH-associated protein 1 (KEAP1) (70). NFE2L2-mediated ferroptosis resistance in HCC cells is achieved by the upregulation of multiple target genes, including well-known NFE2L2-targeted genes like NAD(P)H quinone dehydrogenase 1 (NQO1), GPX4, SLC7A11, and ferritin heavy-chain 1 (FTH1), as well as the newly identified NFE2L2-targeted gene metallothionein 1G (MT1G) (71). MT1G has also been found to be a positive predictor of sorafenib responses in patients with HCC (72). In the context of sorafenib treatment, the upregulation of mRNAs coding for ATP binding cassette subfamily C member 5 (ABCC5) or sigma non-opioid intracellular receptor 1 (SIGMAR1) by NFE2L2 promotes ferroptosis resistance by promoting drug efflux or activating the system xc⁻-GSH-GPX4 pathway in HCC cells (73, 74). Further research is needed to determine whether the expression of ABCC5 or SIGMAR1 is related to the effectiveness of sorafenib treatment in a clinical setting.

Radiotherapy has been used in the treatment of HCC for many years. It not only triggers apoptosis but also induces ferroptosis. In HCC cells, the expression of adiponectin receptor 1 (ADIPOR1) is upregulated and actually constitutes a negative prognostic biomarker after stereotactic body radiation therapy (75). ADIPOR1 is a mediator of resistance to radiotherapy-induced ferroptosis by activating NFE2L2-dependent SLC7A11 expression (75). However, it is not yet clear if adiponectin, the known ligand of ADIPOR1, has a similar effect in inhibiting ferroptosis. ADIPOR1-mediated changes in cell membrane composition may also play a role in impairing lipid metabolism during ferroptosis, independent of adiponectin (76).

Multiple mechanisms have been identified that regulate the expression or activity of NFE2L2, which in turn controls the sensitivity of HCC cells to ferroptosis. As a first example, the long non-coding RNA LINC01134 increases the expression of GPX4 by enhancing NFE2L2 binding to the GPX4 promoter, which in turn inhibits oxaliplatin-induced ferroptosis in HCC cells (77). The activation of the LINC01134-NFE2L2-GPX4 pathway is also associated with oxaliplatin resistance in HCC patients (77). As a second example, the enzyme glutathione S-transferase zeta 1 (GSTZ1), which is involved in phenylalanine catabolism, suppresses NFE2L2 expression and enhances sorafenib-induced ferroptosis in HepG2 and SNU449 cells or in xenograft models (78). The GPX4 inhibitor RSL3 can restore the sensitivity of GSTZ1^−/− HCC cells or Gstz1^−/− mice to sorafenib, suggesting that NFE2L2-mediated GPX4 expression contributes to sorafenib resistance (78). As a third example, quiescin-sulfhydryl oxidase 1 (QSOX1), a disulfide catalyst, enhances sorafenib-induced ferroptosis by inhibiting epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR)-dependent NFE2L2 activation in HCC cells (79). The negative correlation between QSOX1 and NFE2L2 expression in HCC patient tumor tissues further supports the idea that QSOX1 is a negative regulator of antioxidant defense (79). Given that anti-EGFR antibodies like cetuximab can increase the susceptibility of KRAS-mutant colorectal cancer cells to ferroptosis (80), it may be worth testing the combination of cetuximab and sorafenib in inducing ferroptosis in HCC. Finally, the inhibition of several upstream kinases (e.g., TANK binding kinase 1 [TBK1]) or enzymes (e.g., pro-protein convertase subtilisin/kexin type 9 [PCSK9]) can cause NFE2L2 degradation through the SQSTM1-KEAP1/NFE2L2 pathway, thereby promoting ferroptosis in HCC cells (81, 82).

TP53

Tumor protein p53 (TP53) is one of the most eminent tumor suppressors. Approximately 30% of HCC cases are associated with TP53 gene mutations, making it the second-most frequent gene mutation in HCC (83). TP53 is activated in the hepatocytes of individuals with chronic liver disease, which can be caused by viruses, alcohol use, and fat accumulation. Interestingly, in mice lacking the Mdm2 proto-oncogene, causing TP53 accumulation in hepatocytes, TP53 paradoxically promoted HCC development by inducing excessive cell death, chronic inflammation, and a compensatory expansion of hepatic progenitor cells (84). This suggests that TP53 may play an unrecognized role in liver tumorigenesis by selectively sustaining cancer stem cell growth in a cell death-initiated inflammatory tumor microenvironment.

The role of TP53 in ferroptosis is a subject of ongoing research, but most studies suggest that TP53 plays a pro-ferroptotic role in HCC cells by inducing protocadherin beta 14 (PCDHB14) to inhibit the expression of SLC7A11 that is mediated by nuclear factor kappa B (NF-κB) (85) (Fig. 2B). This process is also regulated through the phosphorylation of TP53 on serine 46, which is activated by cell death signals to increase TP53 transcriptional activity. Zinc finger and SCAN domain containing 25 (ZSCAN25, also known as ZNF498) is a transcriptional regulator that can suppress apoptosis and ferroptosis in HepG2 cells by inhibiting serine 46 phosphorylation of TP53 by the complex formed by protein kinase C delta (PRKCD/PKCδ) and tumor protein p53-inducible nuclear protein 1 (TP53INP1) (86). In addition to promoting diethylnitrosamine-induced liver tumorigenesis in mice, higher levels of ZSCAN25 in tumors have been linked to a lower survival rate in HCC patients, suggesting that ZSCAN25 may have an oncogenic effect due to its anti-TP53 activity in apoptosis and ferroptosis (86). Whether targeting ZSCAN25-mediated TP53 inhibition is a potential strategy to restore TP53 tumor suppressor function will require further validation in transgenic animal models.

Glutaminase 2 (GLS2) is a mitochondrial enzyme that is highly expressed in the liver and is responsible for the hydrolysis of glutamine to glutamate in mitochondria. A reduced expression of GLS2 is common in HCC patients, and reducing its expression promotes HCC growth by activating the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (MTOR) pathway, which is a negative regulator of ferroptosis (87). These findings also suggest that TP53 may increase GLS2 expression to promote ferroptosis in HCC cells (87). However, the induced expression of GLS2 not only promotes ferroptosis but also mediates oxidative defense (88). More work is needed to define the exact role of GLS2 in modulating oxidative protection and damage.

Finally, TP53 has been shown to promote the upregulation of phospholipid transfer protein (PLTP), which transfers phospholipids from triglyceride-rich lipoproteins into high density lipoprotein. Mice carrying the P47S variant of TP53 (which cannot undergo phosphorylation of the adjacent serine 46) are highly susceptible to HCC (89). TP53-mediated PLTP upregulation promotes lipid droplet formation and confers resistance to ferroptosis in HepG2 cells with the P47S variant (90). Of note, TP53 does not only act as a transcription factor but also acts outside of the nucleus (91). Thus, TP53 suppresses ferroptosis in HCC by directly inhibiting dipeptidyl peptidase 4 (DPP4)-mediated NOX activation (92). Taken together with previous studies in colorectal cancer (93), these findings suggest that TP53 may exert an anti-ferroptotic role in HCC. It will be important to understand which gene-transactivating and extranuclear effects of TP53 contribute to a probably context-dependent modulation of ferroptosis.

YAP1

Yes1-associated transcriptional regulator (YAP1) is the mammalian equivalent of the Drosophila gene Yorkie (Yki) and plays an important role in the regulation of cell development, growth, repair, and homeostasis. YAP1 acts as a transcriptional coactivator and is regulated by the Hippo signaling pathway. YAP1 dysfunction has been linked to the development and progression of various cancers, including HCC, where it is expressed in approximately 62% of cases (94). The expression of YAP1 in HCC is correlated with a higher serum level of alpha-fetoprotein and poor tumor differentiation (95).

In HCC cells, YAP1 promotes ferroptosis by increasing the expression of TF receptor (TFRC, also known TFR1 or CD71), leading to the accumulation of intracellular free iron and ROS production (Fig. 2C). The O-GlcNAcylation of YAP1, which is a posttranscriptional modification catalyzed by O-linked N-acetylglucosamine (O-GlcNAc), can further enhance the activation of TFRC expression and promote ferroptosis in Bel-7402 and SMMC-7721 HCC cells (96). In contrast, O-GlcNAcylation of the transcription factor c-JUN on serine 73 can suppress ferroptosis by stimulating the synthesis of GSH without affecting phospholipids and Fe²⁺ (97). Thus, targeting O-GlcNAcylation to modulate ferroptosis sensitivity requires consideration of its protein substrates.

Epigenetic mechanism

The modification of RNA by N6-methyladenosine (m6A) is prevalent in eukaryotic messenger RNAs (mRNAs) and influences ferroptosis in HCC cells. The translation of SLC7A11 RNA into protein is facilitated by m6A modification, leading to resistance against ferroptosis (98). The expression of SLC7A11 is suppressed by the core component of the m6A methyltransferase complex, methyltransferase 14, N6-adenosine-methyltransferase subunit (METTL14), which has a tumor-suppressive effect under hypoxia in HCC (99). Conversely, DAZ-associated protein 1 (DAZAP1), which is oncogenic in HCC, can increase resistance to ferroptosis by directly binding to the 3’UTR of SLC7A11 mRNA, thus promoting SLC7A11 protein expression (100). Furthermore, various RNA molecules, including circular RNAs (e.g., CircIL4R), miRNAs (e.g., miR-541-3p), and lncRNAs (e.g., LINC01134, lncFAL, HEPFAL, and GABPB1-AS1), can regulate the sensitivity of HCC cells to ferroptosis by targeting the mRNAs of GPX4, AIFM2, or peroxiredoxin 5 (PRDX5), thus impacting cellular antioxidant capacity (101-104). Collectively, these studies may provide information for the development of ferroptosis-associated RNA drugs.

Protein degradation mechanism

The susceptibility of HCC cells to ferroptosis is influenced by two major intracellular degradation mechanisms: the ubiquitin–proteasome system and autophagy. Tribbles pseudokinase 2 (TRIB2) is a player in liver tumorigenesis, functioning as a molecular adapter that facilitates protein ubiquitination and destabilization. An increase in TRIB2 levels in Bel-7404 and SK-Hep1 HCC cells suppresses ferroptosis by promoting TFRC ubiquitination and reducing labile iron levels through the beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC/βTrCP) pathway (105). The downregulation of centrosomal protein 290 (CEP290) and ubiquitin-like modifier activating enzyme 1 (UBA1) reduces the level of NFE2L2 protein by the ubiquitin–proteasome pathway, leading to ferroptosis in HCC cells (106, 107).

Autophagy, especially selective autophagy, may also contribute to the ferroptotic demise of HCC cells (Fig. 3). As previously mentioned, RAB7A-mediated lipophagy increases lipid availability, thereby enhancing the sensitivity of HCC cells to ferroptosis (29). Activating nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy by erastin, sorafenib, or sulfasalazine can suppress SREBP1-mediated BCAT2 expression in HCC cells, and BCAT2 acts as a suppressor of ferroptosis by increasing intracellular glutamate and subsequent GSH production (108). These findings also establish a connection between protein degradation and the activation of transcription factors that affect lipid homeostasis and antioxidant production. Another example of this process is provided by artesunate-induced ferritinophagy-dependent ferroptosis in HCC cells (109), although the proximal target of artesunate remains elusive.

Figure 3. The role of autophagy in ferroptosis in HCC.

Autophagy is a degradation process that occurs within lysosomes and involves the formation of multiple membrane structures to engulf cellular components such as organelles or proteins. Nonselective autophagy promotes cell survival by recycling and reusing degraded materials to produce energy. Conversely, selective autophagy, including ferritinophagy and lipophagy, can trigger ferroptosis by increasing the availability of free iron and PUFAs. The protein BECN1 can bind to SLC7A11, limiting the production of GSH and the activation of GPX4. On the other hand, PNO1-mediated autophagy can prevent ferroptosis in HCC cells by increasing the synthesis of GSH.

Beclin 1 (BECN1) is a crucial regulator of autophagy that can bind SLC7A11 and inhibit GSH production, thereby suppressing sorafenib-induced ferroptosis (110). HCC cells with a high expression of GABARAPL1 (a member of the LC3 superfamily of proteins required for autophagosome formation) are particularly vulnerable to ferroptosis caused by erastin and sorafenib (111). However, recent research shows that partner of NOB1 homolog (PNO1)-induced autophagy can inhibit sorafenib-induced ferroptosis by promoting GSH biosynthesis in Hep3B and HLE HCC cells (112), highlighting the dual role of autophagy in cell survival and death in the context of ferroptosis stimulation. Further investigation is required to clarify the specific targets degraded by autophagy and how they impact ferroptosis in positive or negative terms.

Cellular uptake mechanism

The process of micropinocytosis, through which molecules dissolved in extracellular fluid cross the cell membrane in small vesicles, has been found to play a significant role in preventing sorafenib-induced ferroptosis in HCC cells (Fig. 4). The activation of the PI3K-Rac family small GTPase 1 (RAC1)-ribonuclease A family member 9 (RNASE9/PAK1) signaling pathway, triggered by mitochondrial damage, is responsible for sorafenib-driven macropinocytosis in HCC cells (113). Amiloride, an inhibitor of micropinocytosis, enhances the capacity of sorafenib in killing HCC cells (113). Indeed, sorafenib-resistant HCC cells are characterized by high levels of macropinocytosis, and suppressing micropinocytosis makes these resistant tumors more responsive to sorafenib treatment (113). Targeting micropinocytosis therefore holds a potential to reduce the development of sorafenib resistance and to improve therapeutic outcomes.

Figure 4. The role of micropinocytosis in ferroptosis in HCC.

Sorafenib can suppress the activity of system xc⁻, reducing cystine uptake and the subsequent synthesis of cysteine and GSH in HCC cells. This is essential for the functioning of GPX4, which helps prevent lipid peroxidation and ferroptosis. However, sorafenib's impact on the mitochondria can trigger a negative feedback mechanism, activating the PI3K-RAC1-RNASE9 pathway. This results in micropinocytosis-mediated uptake of extracellular protein, producing more cysteine to counteract the effects of ferroptosis.

Ferroptosis in HCC tumorigenesis

Inflammation-associated immune suppression is often linked to the development and progression of cancer, and a ferroptosis-induced macrophage polarization has been shown to promote pancreatic cancer tumorigenesis through the use of Pdx1-Cre;Kras^G12D/+;Gpx4^−/− mice (114). Similarly, the loss of hepatocyte-restricted GPX4 in mice does not suppress hepatocellular tumorigenesis (115). Instead, Gpx4 depletion induces ferroptotic death of hepatocytes, which triggers a tumor-suppressive microenvironment characterized by the upregulation of immune checkpoints and the infiltration of myeloid-derived suppressor cells (MDSCs) mediated by high-mobility group box 1 (HMGB1) (115). Thus, ferroptotic damage-mediated inflammation-associated immune suppression may promote the tumorigenesis of HCC. This is also supported by several studies that have shown that iron overload in HCC can drive tumorigenesis, proliferation, and growth (116). Additionally, ferroptosis is related to both acute liver diseases (e.g., carbon tetrachloride-induced liver injury (117)) and chronic liver diseases (e.g., hepatitis B/C (118, 119), non-alcoholic fatty liver disease (120-123), and hereditary hemochromatosis (124)), which can progress to HCC (Fig. 5). The overexpression of the ferroptosis core promoter ACSL4 in HCC patients has been linked to poor patient outcomes (31). The depletion of ACSL4 in HCC cells can effectively inhibit cell proliferation by disrupting the c-Myc-SREBP1 pathway (31). Furthermore, lipid peroxidation-derived DNA adducts, such as γ-OHPdG, have been shown to promote hepatocarcinogenesis in mice (125). Therefore, reducing exposure to risk factors that cause ferroptotic damage could help prevent HCC.

Figure 5. The dual roles of ferroptosis in HCC.

On the one hand, ferroptotic damage has been linked to a number of acute or chronic liver diseases that can lead to HCC. On the other hand, inducing ferroptosis through chemotherapy, immunotherapy (anti-PDCD/PD-1 or anti-CD274/PD-L1 antibodies) or radiotherapy can be effective in suppressing tumor growth in established HCC.

Ferroptosis in HCC therapy

The induction of ferroptosis may constitute a strategy for combating established HCC (Fig. 5). There are several compounds and clinical drugs that have been used to trigger ferroptosis in HCC (Table 2). As described above, classical inducers, such as erastin and RSL3, inhibit the system xc⁻-GSH-GPX4 pathway. Among ferroptosis-inducing drugs, sorafenib is most commonly used to treat HCC. Sorafenib is an oral multi-kinase inhibitor used in the treatment of advanced renal cell carcinoma, liver cancer, and thyroid cancer. Sorafenib was initially characterized as an apoptosis inducer, and this activity was attributed to its action on oncogenic kinases (126). The mechanism of ferroptosis induced by sorafenib is not mediated by kinase inhibition (127). Indeed, high doses of sorafenib (10 μM) may induce ferroptosis by directly inhibiting SLC7A11, although this is controversial (128). Regardless, cancer cells display varying degrees of sensitivity to ferroptosis induction upon the inhibition of system xc⁻, as the transsulfuration pathway can bypass system xc⁻ by synthesizing cysteine from methionine to maintain GSH synthesis (129).

Table 2.

Examples of ferroptosis inducers and inhibitors

Name	Mode of action	Application	References
Ferroptosis inducers
Abemaciclib	Inhibits CDK4/6	Preclinical and clinical	(130)
Alkaloid trigonelline	Inhibits NFE2L2	Preclinical	(70)
Anthracyclins	Increases PUFA	Preclinical	(149)
Artesunate	Induces ferritinophagy	Preclinical and clinical	(109)
Disulfiram	Induces mitochondrial damage	Preclinical and clinical	(150)
Erastin	Inhibits SLC7A11	Preclinical	(70)
Haloperidol	Inhibits SIGMAR1	Preclinical	(74)
Oxaliplatin	Induces ROS production	Preclinical and clinical	(77)
RSL3	Inhibits GPX4	Preclinical	(21)
Saponin formosanin C	Induces ferritinophagy	Preclinical	(18)
Solasonine	Inhibits GPX4	Preclinical	(151)
Sorafenib	Inhibits SLC7A11	Preclinical and clinical	(128)
Tiliroside	Inhibits TBK1 and induces NFE2L2 degradation	Preclinical	(82)
Vanadium -iIron -oOxide	Inhibits GPX4	Preclinical	(152)
ZZW-115	Inhibits NUPR1	Preclinical	(59)
Ferroptosis inhibitors
Deferoxamine	Iron chelator	Preclinical and clinical	(153)
Deferasirox	Iron chelator	Preclinical and clinical	(154)
Ferrostatin-1	Free radical-scavenging antioxidant	Preclinical	(153)
Liproxstatin-1	Free radical-scavenging antioxidant	Preclinical	(153)
Baicalein	Inhibits ALOX	Preclinical and clinical	(65)
Zileuton	Inhibits ALOX	Preclinical and clinical	(66)

Abemaciclib, a cyclin-dependent kinase (CDK) inhibitor selective for CDK4 and CDK6, has been clinically approved for the treatment of advanced metastatic breast cancer. Theoretically, the combination of abemaciclib and sorafenib has the potential to improve patient outcomes in HCC by inducing synthetic lethality. However, phosphoseryl-TRNA kinase (PSTK) has been identified as a mediator of resistance to abemaciclib and sorafenib treatment, inhibiting ferroptosis induction in HCC through the activation of GPX4 (130).

Oxaliplatin, a third-generation platinum drug used as first-line chemotherapy for colorectal cancer, can mediate resistance to ferroptosis induction in HCC through the expression of NFE2L2-mediated GPX4 (77). Radiotherapy has also been reported to induce ferroptosis in HCC cells by inhibiting SLC7A11 (131) or SLC1A5 (132). The use of engineered Man@pSiNPs-erastin has the potential to boost the effectiveness of immune checkpoint inhibitors (anti-PDCD/PD-1 or anti-CD274/PD-L1 antibodies) in an HCC model by suppressing pro-tumor polarization of macrophages (133). In addition, inhibitors of NFE2L2, such as alkaloid trigonelline, are expected to enhance the sensitivity of HCC cells to ferroptosis in cell cultures and in mouse models (70).

There are also natural products, metal-containing drugs, and nanoparticles that have been identified as potential ferroptosis inducers in HCC cells (examples are provided in Table 2). These findings have provided valuable pharmacological tools for investigating the signals and mechanisms of ferroptosis. However, they have also raised concerns about the specificity of ferroptosis-inducing agents.

Monitoring ferroptosis sensitivity

Non-invasive methods that measure the mechanisms and dynamics of ferroptosis would be extremely useful for understanding the diversity of HCC patients with respect to ferroptosis-inducing treatments, including drug therapy with sorafenib, alone or in combination with other drugs.

Effective monitoring of the response to drug-induced ferroptosis in cancer patients requires the use of sensitive and specific biomarkers, preferentially in serum or plasma. Mass spectrometry can be used to analyze changes in serum iron or lipid metabolites, while enzyme-linked immunosorbent assays (ELISAs) of damage-associated molecular patterns (DAMPs) can offer a quick, simple, and cost-effective way to assess ferroptosis. DAMPs, such as HMGB1 (134), are molecules released by dead or dying cells, irrespective of the specific death modality. Ferroptotic cells specifically release decorin (DCN) (135), a proteoglycan from the small leucine-rich proteoglycan family. In healthy livers, small amounts of decorin are found around central veins and in portal tracts, but during fibrogenesis, its levels significantly increase in connective tissue septa. Studies in mice have shown that decorin can inhibit liver carcinogenesis (136, 137). Nonetheless, pending further investigation, an elevation in serum DCN concentrations might indicate a therapeutically relevant sorafenib-induced ferroptosis response. Ferroptotic cells also release prostaglandin E2 (PGE2), which may inhibit the antitumor activity of immune cells (138). Whether the quantitation of circulating PGE2 provides information on the efficacy of sorafenib has not been determined.

Iron probes are valuable tools for measuring iron levels in different biological samples. Chromogenic methods using iron probes provide a convenient method for the simultaneous measurement of Fe²⁺ and Fe³⁺ (139). Fluorescent iron probes such as FerroFarRed, Calcein-AM, and FRET Iron Probe 1 are also widely used to analyze the dynamic changes of iron levels during ferroptosis. Lipid hydroperoxides are the major product of peroxidation reactions and can be measured directly by high-performance liquid chromatography (HPLC) with chemiluminescent detection. Furthermore, combining C11-BODIPY(591/581) staining with flow cytometry measurements can provide multiparametric information on intracellular oxidative stress damage in single cells during ferroptosis. High-throughput lipidomics can comprehensively assess abnormal lipid metabolism in ferroptosis.

Finally, immunohistochemistry analyses of key ferroptosis regulators, such as ACSL4, GPX4, SLC7A11, and TFRC, in surgically resected liver tumor samples can provide valuable insights into treatment response. In particular, ACSL4 has been shown to predict the prognosis of sorafenib-treated HCC patients, especially those experiencing sorafenib-mediated ferroptosis (140).

Clinical application of a ferroptosis scoring system in HCC

Bioinformatics has revealed a potential clinical connection between the ferroptosis-related genes and HCC patients. A prognostic scoring system has been established based on the expression of 25 ferroptosis-related genes in public TCGA, CGGA, and GEO databases. A consensus clustering analysis of 371 HCC cancer samples resulted in 3 independent clusters (141). Cluster 3 was found to have higher expression levels, an unfavorable prognosis, and higher histological tumor stage and grade compared to clusters 1 and 2. The expression of HSPA5, EMC2, SLC7A11, HSPB1, GPX4, FANCD2, CISD1, FDFT1, SLC1A5, TFRC, RPL8, DPP4, CS, CARS1, ATP5MC3, ALOX15, ACSL4, and ATL1 was significantly increased in HCC tissue compared to normal liver tissue (141). Conversely, the expression of NFE2L2, MT1G, SAT1, and GLS2 was decreased in HCC tissue compared to normal liver tissue (141). Among these genes, the increased expression of SLC7A11, SLC1A5, TFRC, RPL8, and CARS1 was associated with unfavorable overall survival in HCC patients (141). Another study assessed 374 HCC tumor samples using 214 ferroptosis-related genes from the FerrDb database (142). This study identified a four-gene signature for overall survival in HCC, consisting of GPX2, MT3, PRDX1, and SRXN1. PRDX1 was found to be the hub gene of the prognosis model and had high expression in HCC tumor tissue (142). On the other hand, researchers assessed 104 ferroptosis- and iron metabolism-related genes and found that a prognosis model constructed with four genes (ABCB6, FLVCR1, SLC48A1, and SLC7A11) independently predicted the prognosis of HCC patients with superior accuracy (143). The median value of the ferroptosis score was used to classify HCC patients into high- and low-ferroptosis score groups. HCC patients with high-ferroptosis scores exhibited a stronger immunosuppressive tumor microenvironment (143). More recently, 10 ferroptosis-related genes (UGT1A6, ATP6V1C1, MAFG, NUDCD1, PPP1R1A, TSKU, CTSB, AIFM2, CTSA, and CTNND2) were found to be posttranscriptionally regulated by RNA modifications in HCC (56).

Collectively, ever more sophisticated in silico analyses of big data could lead to the development of a predictive and prognostic ferroptosis scoring system applicable to HCC patients. To achieve this goal, it will be crucial to increase sample size, to gather more comprehensive clinical information on each HCC case, and to validate all results in independent cohorts. Additionally, it will be interesting to monitor ferroptosis-related molecular changes over time to understand their prognostic and predictive value for treatment responses and the subsequent resistance-associated relapse.

Conclusion and perspective

The discovery of ferroptosis as a type of non-apoptotic cell death has sparked numerous preclinical studies examining its contribution to chemotherapy, radiotherapy, or immunotherapy responses in HCC. Ferroptosis exhibits different in vitro and in vivo features than the well-studied apoptosis (Table 3). The effect of ferroptosis on liver cancer progression is complex and can vary at different stages of the tumor. Further research into the factors that influence the antitumor immune response in ferroptosis is crucial for developing new treatments and combination therapies. Of note, HCC patients often exhibit abnormal iron absorption and lipid metabolism disorders, which play a significant role in regulating ferroptosis. An aberrant expression of several ferroptosis regulators is also associated with poor HCC prognosis (Table 1).

Table 3.

Differences between ferroptosis and apoptosis

	Ferroptosis	Apoptosis
Morphological features	Enlarged and swollen cells with reduced mitochondrial volume and plasma membrane rupture	Cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation
Biochemical properties	Iron accumulation and lipid peroxidation	Caspases activation in many cases
Immunological features	Pro-inflammatory in many cases	Anti-inflammatory in many cases
Key positive regulators	ACSL4, POR, and ALOX	CASP3, CASP7, CASP8, and CASP9
Key negative regulators	SLC7A11, GPX4, AIFM2, DHODH, and NFE2L2	BCL2, BCL2L1/BCLXL, and MCL1
Autophagy response	Increased autophagy promotes ferroptosis	Increased autophagy inhibits apoptosis
In vivo intervention	Ferrostatin-1 or liproxstatin-1 inhibits ferroptotic damage in various tissues	Caspase inhibitor Z-VAD-FMK alleviates apoptotic damage in various tissues
Biomarkers	ACSL4, DCN, PTGS2/COX2, and TFRC	Cleaved-CASP3, Cleaved-CAPS8, and Cleaved-PARP

Despite progress in the area of ferroptosis, translating ferroptosis research into effective treatments for patients with HCC remains a challenge. One obstacle is that key regulators of ferroptosis, such as GPX4 and SLC7A11, are widely expressed throughout the body, making it difficult to target them without causing side effects. Additionally, multiple antioxidant systems can inhibit ferroptosis, meaning that targeting one single pathway may not be sufficient for a lasting response. Although the mechanism of ferroptosis is highly context-dependent, in many cases the deletion of the key ferroptosis promoters ACSL4 or POR can demonstrate ferroptosis in vivo and in vitro. A further challenge is the absence of confirmed biomarkers that can differentiate ferroptotic from non-ferroptotic cell death, making it difficult to determine the effectiveness of potential treatments. Combined analysis of specific lipid peroxide products and DAMPs (e.g., DCN) in serum may be a relatively sensitive and specific method for detecting ferroptosis in vivo.

To address these obstacles, gaining a deeper understanding of the signals, mechanisms, and modulations of ferroptosis in response to specific agents or combinations is crucial. Understanding the sensitivity and resistance of ferroptosis is a key objective. This requires the development of drugs specifically designed to induce ferroptosis in cancer cells while avoiding normal cells. Furthermore, the identification of accurate and easily accessible biomarkers will be instrumental in developing personalized ferroptotic therapies.