4.1.1 Nutrient availability

Both normal and tumor cells require sufficient nutrients and energy to survive. However, the metabolic requirements of cancer cells and nutrient availability obtained by tumors from the environment may differ from those of normal cells. Nutrients available to tumors can directly affect not only tumor survival and proliferation, but also other genes and metabolic pathways [53]. Thus, by limiting the nutrient availability required by tumors in the environment and the intrinsic ability of cells to acquire and efficiently utilize these nutrients, it may be possible to modify tumor cell resistance to cell death, such as ferroptosis. Some intracellular nutrients (trace elements and vitamin E, etc.) closely related to the cellular antioxidant system, as well as exogenous nutrients such as glucose and amino acids, have been reported to be involved in the regulation of ferroptosis-related pathways [54].

Environmental stress from limiting or supplying exogenous nutrients such as glucose, amino acids, or fatty acids may sensitize tumor cells to ferroptosis. Glucose depletion inhibits the sensitivity of human pancreatic ductal carcinoma cell (PDAC) to ferroptosis. Glucose transporter SLC2A1-mediated glucose uptake promotes glycolysis, thereby promoting pyruvate oxidation and stimulating fatty acid synthesis, ultimately promoting lipid peroxidation-dependent ferroptosis. As a driver of ferroptosis resistance, SLC2A1-dependent pyruvate dehydrogenase kinase 4 (PDK4) promotes glucose-dependent ferroptosis resistance by inhibiting pyruvate dehydrogenase [5]. The cystine/glutamate antiporter SLC7A11 could transport cystine to promote the biosynthesis of GSH and maintain the redox balance, resulting in protection from ferroptotic cell death [55]. However, SLC7A11^high cancer cells are highly dependent on glucose or glutamine for survival and are exquisitely sensitive to glucose or glutamine starvation-induced cell death [56-59]. In SLC7A11^high cancer cells, a large amount of imported cystine is likely toxic due to its low solubility, and this imported cystine is quickly reduced to more soluble cysteine in the cytosol in a manner dependent on NADPH supplied from glucose via the pentose phosphate pathway [60]. Limiting glucose availability in SLC7A11^high cancer cells results in NADPH depletion and disulfide stress-related cell death [59]. In addition, high glutamate export drives more glutamine uptake to activate glutaminase for glutamate replenishment, making cells more sensitive to glutamine starvation [60]. These studies indicated the complexity of nutrient availability on cell fate determination and suggested that the relationship between nutrient limitation and ferroptosis needs to be further explored. In addition to glucose and glutamine, Recent evidence suggests that other nutrients are closely related to ferroptosis. Non-small cell lung cancer (NSCLC) cells with high NRF2 expression protect themselves from cystine starvation-induced ferroptosis by enhancing γ-glutamyl-peptide synthesis to limit the accumulation of glutamate via a noncanonical activity of glutamate-cysteine ligase catalytic subunit (GCLC) [61]. Exogenous fatty acid levels affect ferroptosis susceptibility in a “double-edged” manner. Enrichment of PUFAs in the plasma membrane predisposes tumor cells to ferroptosis. Dietary intake of the PUFA dihomo-gamma-linolenic acid (DGLA; 20:3n−6) induces ferroptosis in Caenorhabditis elegans and human cells [62]. Moreover, dietary supplementation with n-3 and n-6 PUFAs selectively induces ferroptosis in tumor cells in an acidic environment [6]. In contrast to PUFAs, however, administration of MUFAs make cells entering a ferroptosis-resistant cell state [63]. Exogenous MUFAs are converted to MUFA-PLs in an acyl-CoA synthetase long-chain family member 3 (ACSL3)-dependent manner, then reducing PUFA incorporation into phospholipids, replacing PUFAs in membrane lipids, hindering lipid ROS accumulation and inhibiting ferroptosis. Furthermore, exogenous MUFAs also protect cells from apoptotic lipotoxicity caused by saturated fatty acid accumulation in an ACSL3-independent manner.

Some intracellular nutrients are also involved in the regulation of ferroptosis. Regulation of cellular iron levels by altering iron storage and export is critical for ferroptosis. Inhibition of mitochondrial protein frataxin (FXN) expression significantly enhances erastin-induced ferroptosis by accelerating the accumulation of free iron and lipid peroxidation [64]. In terms of regulating iron transport, prominin2 could inhibit ferroptosis by promoting the formation of ferritin-containing multivesicular bodies (MVBs) and exosomes to transport iron out of cells [65]. In addition, iron ions may coregulate iron homeostasis metabolism with other metal ions to cope with the stress brought by iron loading. The FDA-approved anti-rheumatoid arthritis drug auranofin (AUR) is a small molecule compound containing aurum that upregulates hepcidin by activating the NF-κB/IL-6/JAK-STAT signaling pathway to reduce iron overload. High-dose AUR inhibits the thioredoxin reductase (TXNRD) family of enzymes and leads to lipid peroxidation and ferroptosis [66]. Arsenite can trigger major ultrastructural damage in mitochondria and induce oxidative stress, which leads to testicular cell death by inducing ferroptosis [67]. Abundance of the trace element selenium also affects ferroptosis sensitivity [68]. GPX4 requires selenium for its biosynthesis, and dietary selenium supplementation promotes ferroptosis resistance by upregulating the abundance and activity of GPX4 [69]. Selenium-GPX4 could protect follicular helper T (TFH) cells from ferroptosis. In GPX4-deficient immunized mice, the number of TFH cells was significantly reduced and unable to produce high-affinity protective antibodies. Enhancing the expression of GPX4 in T cells by dietary selenium supplementation can enhance the number of TFH cells and improve the antibody immune response [70]. Dietary vitamin E supplementation is also effective in preventing ferroptosis. GPX4 and vitamin E synergistically maintain lipid redox balance and prevent ferroptosis in hematopoietic stem and progenitor cells [71].

4.1.2 Heat stress and cold stress

Cells are continuously exposed to various environmental signal fluctuations, including fluctuations in temperature and pH value, and so on. Maintaining a steady temperature is vital for life. A sudden temperature rise destroys important cell structures and interferes with basic cell functions [72]. In response to heat stress, cells activate heat shock proteins (HSPs). HSPs have a complex protective mechanism that prevents nonspecific protein aggregation and helps the protein maintain its inherent structure. Heat shock factors (HSFs) are transcription factors that regulate the synthesis of HSPs. Heat shock protein beta-1 (HSPB1) is a small heat shock protein (sHSP), also called mouse Hsp25 or human HSP27. Studies have found that activation of the heat shock factor 1 (HSF1)-HSPB1 pathway can negatively regulate erastin-induced ferroptosis [73]. Protein kinase C (PKC)-mediated HSPB1 phosphorylation negatively regulates ferroptosis by inhibiting iron uptake by cells and the production of lipid ROS. Gene knockdown or drug inhibition of the HSF1-HSPB1 pathway significantly improves the anticancer activity of erastin in vivo and in vitro, providing a basis for the combinational therapy targeting HSPB1 and ferroptosis for tumor treatment [73]. HSF1 also protects cardiomyocytes from palmitic acid-induced ferroptosis by maintaining cellular iron homeostasis and restoring GPX4 expression [74]. In addition, heat shock in plant cells causes ROS to accumulate and consumes GSH, which in turn triggers iron-dependent oxidative cell death similar to the ferroptosis in animal cells [75].

The body temperature of a warm-blooded animal is kept approximately at 37°C even in a cold environment. However, it is difficult for most cells to generate heat to maintain the intracellular temperature. Dramatic changes in the extracellular environment can usually cause severe damage to cells, leading to cell death. Among them, the precise molecular regulation mechanism of cold stress on the novel death mode-ferroptosis is still unclear. Cold stress-induced ferroptosis can be observed in multiple tumor cell lines such as A549, HepG2, and HT-1080. Apoptosis signal-regulating kinase 1 (ASK1) gene-deficient mice are sensitive to cold stress [76], and severe cold stress can activate the p38 mitogen-activated protein kinase (MAPK) pathway [77]. These findings suggest that ASK1 and MAPK signals may be involved in the cold stress response. Later, in 2017 a team from Japan found that sustained severe cold stress induces ferroptosis through the accumulation of lipid peroxides [78]. To cope with continuous exposure to an extremely low-temperature environment, lipid peroxides accumulate in a time-dependent manner and activate downstream ASK1-p38 MAPK pathways, thereby inducing ferroptosis. Ferroptosis inhibitors such as Fer-1, iron chelator deferoxamine (DFX), and p38 inhibitors are considered potential therapeutic drugs to prevent cold-induced damage. The link between cold stress and tumor growth was discovered more than 40 years ago [79], and given the discovery of the association between cold stress and ferroptosis, we need to focus on the regulation of cold stress-induced ferroptosis and thus on tumor growth in the future.

4.1.3 Hypoxia and metabolic acidosis

Hypoxia is one of the basic characteristics of solid malignant tumors and is closely related to tumor angiogenesis, metastasis, and drug resistance [80]. Tumor cells adapt to the anaerobic microenvironment through the release of tumor hypoxia-inducible factors (HIFs) [80]. The effect of hypoxia on ferroptosis is context-dependent. On the one hand, hypoxia reduces intracellular free iron by increasing ferritin expression to protect cells from ferroptosis [81]. As a hypoxia-responsive protein, carbonic anhydrase 9 (CA9) expression is significantly higher under hypoxia and correlates with increased catalytic Fe (II) in malignant mesothelioma (MM). CA9 inhibitor decreased (ferritin heavy chain/ferritin light chain) FTH/FTL and induced ferroptosis [82]. In this regard, reoxygenation may be an effective strategy to activate ferroptosis. Indeed, an Fe (VI)-based nano-oxygen release system activated by ultrasound can overcome the resistance of tumor cells to doxorubicin (DOX) induced by the hypoxic microenvironment by synergistically inducing apoptosis and ferroptosis in solid tumors [83]. The ferrate released by this system can effectively react with water and high levels of hydrogen peroxide and GSH in tumor cells to achieve tumor microenvironment-independent reoxygenation and GSH depletion. The metabolism of exogenous iron delivered via nanomedicine will initiate Fenton reactions, leading to the excessive production of ROS and iron-dependent cell death. GSH depletion inactivates GPX4, inhibits the reduction of lipid peroxides, thereby enhancing ferroptosis [83]. This evidence suggests that targeted induction of ferroptosis is a therapeutic approach for some hard-to-treat cancers. On the other hand, HIF signaling enhances lipid peroxidation to induce ferroptosis. Clear-cell carcinomas (CCCs) are highly aggressive malignancies distinguished by aberrant lipid and glycogen accumulation. CCCs are refractory to several anticancer therapies but show intrinsic vulnerability to ferroptosis in a hypoxia-dependent manner [8]. In renal CCCs, hypoxia-inducible factor 2 subunit α (HIF-2α) selectively enriches polyunsaturated lipids, the rate-limiting substrates for lipid peroxidation, by activating the expression of hypoxia-inducible, lipid droplet-associated protein (HILPDA). Additionally, autophagy-mediated degradation of aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL) (the core circadian clock protein) promotes the expression of egl nine homolog 2 (EGLN2) to destabilize hypoxia-inducible factor 1 subunit α (HIF-1α) and ultimately promote lipid peroxidation and ferroptosis [84].

Due to the poor oxygen perfusion and enhanced aerobic glycolysis present under hypoxic conditions, the overall tumor tissue is in an acidic microenvironment [85]. Tumor cells with higher glycolysis and acid resistance have more robust growth advantages. Accumulation of n-3 and n-6 PUFAs is toxic to acid-adapted cancer cells [6]. Ferroptosis is promoted when acidic cancer cells fail to buffer the enhanced uptake of PUFAs and expose themselves to the deleterious effects of lipid peroxidation. Most importantly, this dietary PUFAs supplementation has little effect on normal pH tissue cells. In addition, the ability of transferrin to bind iron is highly dependent on changes in pH value. At pH~7.4, iron effectively binds to transferrin outside cells, and when iron is delivered to acidic endosomes (pH~5.5) through receptor-mediated endocytosis, iron is dissociated from transferrin [28]. Currently, the exact molecular mechanism between ferroptosis and acidosis is unknown. Given the close relationship between iron and acidic environments, the effects of stress caused by acidosis on ferroptosis need to be further explored.

4.2.1 Energy stress

Normal cells require sufficient energy to survive. Under pathological conditions, stress-induced metabolic changing triggers several adaptive responses to restore dynamic homeostasis and to maintain cellular function. One type of metabolic stress is energy stress, which is characterized by the consumption of intracellular ATP and a corresponding increase in intracellular adenosine monophosphate (AMP) levels. Energy stress induces an essential adaptive response mediated by AMP-activated protein kinase (AMPK), the cellular energy state sensor [86,87]. After AMPK is activated, ATP anabolism is inhibited to promote ATP catabolism, thereby restoring energy balance and maintaining cell survival. When facing long-term and excessive energy stress, ATP is excessively consumed, and this eventually induces apoptosis [86,87]. In a renal ischemia-reperfusion injury model, AMPK activation was found to inhibit ferroptosis, but this inhibition did not involve autophagy, the mechanistic target of rapamycin complex 1 (mTORC1) signaling, cystine uptake, or iron metabolism regulation [7]. Energy stress activates AMPK to phosphorylate and inactivate acetyl-CoA carboxylase (ACC), reducing the biosynthesis of PUFAs and other fatty acids, and ultimately inhibiting ferroptosis [7]. Liver kinase B1 (LKB1) is the main upstream kinase that activates AMPK in response to energy stress, and it is also one of the most common mutated tumor suppressors in NSCLC and cervical cancer [88]. Similarly, LKB1-AMPK axis phosphorylates ACC1 to negatively regulates fatty acid synthesis, protecting cells from lipid hydroperoxide accumulation and ferroptosis [89]. However, AMPK not only plays a tumor-promoting role by promoting the survival of tumor cells under metabolic or energy stress, but it can also exert a tumor-suppressing effect. AMPK mediates the phosphorylation of Beclin 1 (BECN1) to form a BECN1-SLC7A11 complex, directly block SLC7A11, and promotes ferroptosis through lipid peroxidation [90]. In subcutaneous and orthotopic tumor mouse models, genetically and pharmacologically activated BECN1 pathways increase ferroptosis. Hydroxycarboxylic acid receptor 1 (HCAR1) and monocarboxylate transporter 1 (MCT1) are both lactate transporters that mediate lactate absorption and promote ATP production to inactivate AMPK. In lactate-rich liver cancer cells, HCAR1/MCT1-mediated AMPK inactivation leads to the upregulation of sterol regulatory element-binding protein 1 (SREBP1) and downstream stearoyl-coenzyme A (CoA) desaturase-1 (SCD1), which enhance the production of anti-ferroptosis monounsaturated fatty acids [91]. These findings highlight that AMPK-mediated energy stress has complicated and cell type- or disease-dependent regulatory effects on ferroptosis.

4.2.2 ER stress

Under hypoxia, glucose deficiency, calcium imbalance, oxidative stress, injury, and other stress conditions, protein processing is affected, unfolded or misfolded proteins accumulate in the ER, and the unfolded protein response (UPR) occurs, causing ER stress [92,92]. The ER is the source of lipids for most membranes of other organelles and contains more than half of all the lipid bilayers in any given cell. Causes of ER stress include redox imbalance and lipid peroxidation. Upregulation of ER stress markers such as ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1), activating transcription factor 4 (ATF4), and phosphorylation of eukaryotic initiation factor 2α (eIF2α) can be observed during ferroptosis [94]. Indeed, Erastin and sorafenib, two reported ferroptosis agonists, induce ER stress by upregulating the ER stress response gene CHAC1 [55]. The heat shock protein family A (Hsp70) member 5 (HSPA5, also known as GRP78) is a member of the heat shock family, and it is mainly located in the ER or mitochondria. ATF4 upregulates HSPA5 in PDAC cells, which directly inhibits the degradation of GPX4 and inhibits the lipid peroxidation process of ferroptosis [95]. Genetic inhibition of the HSPA5-GPX4 pathway enhances the anticancer activity of gemcitabine by inducing ferroptosis [96]. The above evidence suggest that ER stress activation plays a negative effect on ferroptosis induction. Therefore, the combined use of ferroptosis inducers (FINs) and ER stress inhibitors may have great therapeutic significance for cancer treatments.

4.2.3 Oncogenic stress

Tumorigenesis is a multi-step and complex process with dysregulated oncogenic pathways involved. Recent evidence suggests that the oncogenic stress induced by hyperactivation of oncogenes or loss of suppressors in some cases is linked to ferroptosis that is associated with oncogenic transformation, tumor growth, and therapy response.

Mutant RAS is the first described oncogene that interplays with ferroptosis. Erastin and RSL3, the two classical FINs, were reported to have selective lethality against tumor cells expressing mutant RAS [15,17]. Inhibition KRAS or its downstream RAS-RAF-MEK-ERK pathway reverses the antitumor effect of erastin [16]. Moreover, KRAS-mutant lung adenocarcinoma cells are vulnerable to SLC7A11 inhibition [97]. These preclinical evidence supports the important interaction between KRAS mutation and ferroptosis, revealing a promising therapeutic strategy against KRAS-mutant tumors. Nevertheless, sensitivity profiling in 177 cancer cell lines revealed ferroptosis can be triggered by erastin regardless of the mutational status of RAS [29]. In contrast, ectopic expression of NRAS12V, KRAS12V, or HRAS12V protects RMS13 rhabdomyosarcoma cells from oxidative stress-induced ferroptotic death, suggesting the precise role of KRAS mutation in modulating ferroptosis is complex and requires further investigation. Notably, KRAS is not the only oncogenic signaling involved in regulating ferroptosis. It was reported that hyperactivation of several oncogenic signaling, including epidermal growth factor receptor (EGFR) pathway, Yes-associated protein (YAP) pathway, and c-Myb (a proto-oncogene protein) pathway, may elevate lipid ROS production and render cancer cells extremely susceptible to ferroptosis. EGFR-mutation sensitized NSCLC cells to ferroptosis in a MAPK signaling-dependent manner [98]. Oncogenic activation of YAP signaling sensitizes HCC cells to ferroptosis via arachidonate lipoxygenase 3 (ALOXE3) mediated lipid peroxidation accumulation [99]. Overexpression of c-Myb sensitizes gastric cancer (GC) cells to ferroptosis via upregulating cysteine dioxygenase type 1 (CDO1) expression and downregulating GPX4 expression [100]. However, hyperactive mutation of phosphatidylinositol-3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signaling protects cancer cells from oxidative stress and suppresses ferroptosis via SREBP-mediated lipogenesis [9].

In addition to oncogenes, ferroptosis also crosstalk with bona fide tumor suppressors. Tumor suppressor p53 is spontaneously mutated or deleted in most human cancers. Despite the general important role of p53 in DNA repair, cell-cycle arrest, senescence, and apoptosis, it has been demonstrated that p53 act as a double-edged sword in ferroptosis. On the one hand, p53 enhances ferroptosis by inhibiting the expression of SLC7A11 or promoting the expression of spermidine/spermine N1-acetyltransferase (SAT1) and glutaminase 2 (GLS2). On the other hand, p53 inhibits ferroptosis by inhibiting dipeptidyl peptidase 4 (DPP4) activity or inducing cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21 expression [101]. Notably, acetylation differentially regulates p53-mediated cell-cycle arrest, senescence, apoptosis, and ferroptosis. The acetylation-defective mutation p53^3KR (K117R + K161R + K162R) lost the ability to induce cell cycle arrest, senescence, and apoptosis, but retains the ability to promote ferroptosis and suppress tumor formation [44]. Loss of an additional acetylation site at K98 (namely p53^4KR) abolish its ability to induce ferroptosis, as well as its remaining tumor suppression activity [102], indicating ferroptosis contributes to p53-mediated tumor suppression. Like p53, tumor suppressor BRCA1-associated protein 1 (BAP1) is a tumor suppressor with frequent mutation or deletion in human cancers. Inactivation of BAP1 in cancer cells results in evaluated SLC7A11 expression, increased cystine uptake and GSH synthesis, and enhanced ferroptosis resistance, which contributes to tumor development. The mechanism how BAP1 suppresses SLC7A11 expression may partially attribute to the deubiquitinating H2A ubiquitination (H2Aub) on SLC7A11 [103]. Merlin, encoded by the neurofibromatosis type 2 gene, is a tumor suppressor frequently inactivated in MM. Genetic inactivation of Merlin renders MM cancer cells more sensitive to ferroptosis by promoting YAP activity, suggesting the involvement of Merlin-YAP signaling in dictating ferroptotic death [104].

Collectively, the above examples illustrate the context-dependent role of dysregulated oncogenic pathways in regulating ferroptosis. It is important to note that individual oncogene or tumor suppressors may play dual role in the control of ferroptotic death due to the tumor heterogeneity. More work is needed toward understanding the ferroptosis regulatory network so that optimal therapeutic approaches can be identified for future drug discovery efforts.

4.2.4 Genomic stress

The genome integrity is critical for the passage of genetic materials, and genomic DNA can be damaged by a variety of exogenous and endogenous sources such as replication errors, chemically induced adducts, ultraviolet (UV) induced damage, and single-strand (SSB) or double-strand (DSB) breaks caused by ionizing radiation (IR) or chemical reactions [105]. We refer to the genomic response induced by the above threats as genomic stress. In response to these threats, eukaryotes have evolved the DNA Damage Response (DDR) to sense DNA damage, repair DNA and process cells with damaged DNA to maintain genome integrity, and, when appropriate, execute cell death programs to remove unrepaired cells [106]. When cells are deficient in DDR and cannot repair damaged DNA, it can lead to genomic instability, one of the fundamental hallmarks of cancer [107]. Many DDR components including DNA damage sensor, signaling sensor, or downstream effector are involved in the regulation of ferroptosis [106]. Focusing on this interaction between DDR-related genomic stress and ferroptosis, a new type of cell death, is crucial in the study of cancer mechanisms.

Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-Related (ATR) are essential for DDR and maintenance of genome stability as kinases that sense DNA damage. Pharmacological or genetic inhibition of ATM protects a variety of cancer cells from ferroptosis induced by cystine deprivation or erastin. However, ATM inhibition rescues ferroptosis not through the canonical DNA damage pathway (ATM-P53/Chk2) but by increasing the expression of iron regulators involved in iron storage (ferritin heavy and light chains, FTH1 and FTL) and export (ferroportin, FPN1). Furthermore, ATM inhibition enhanced nuclear translocation of metal-regulated transcription factor 1 (MTF1), and genetic deletion of MTF-1 abolished ATM regulation of iron regulatory elements and resensitized cells to ferroptosis [108]. Zalcitabine induces the degradation of TFAM (transcription factor A, mitochondrial), which in turn leads to mitochondrial DNA stress and oxidative DNA damage. Mechanistically, Zalcitabine activates the stimulator of interferon response cGAMP interactor 1 (STING1/TMEM173)-mediated DNA sensing pathway leading to macroautophagy/autophagy-dependent ferroptotic cell death via lipid peroxidation [109].

In the DDR pathway, one of the key targets of ATM/ATR is p53 [106]. As an effector of DDR, p53 can respond to DNA damage, respond to oncogenic signals, and promote apoptosis or senescence. p53 can trigger protective pro-survival responses, such as temporary cell cycle arrest, DNA repair, or production of antioxidant proteins that maintain genome integrity and repair damaged cells [110]. The specific molecular regulation mechanism of DDR effector p53 on ferroptosis has been summarized in detail in the section “Oncogenic stress” and will not be repeated here.