Cells can die in two separate ways: accidental cell death (ACD) or regulated cell death (RCD) [129]. RCD is a highly controlled and regulated process that includes signaling cascades, and it is required for tissue homeostasis so that organisms can regulate cell proliferation and cell death, and inhibit tumor growth [130,131]. RCD is common in diseases ranging from neurodegenerative diseases to cancers. The earliest discovered form of RCD, in 1972 by John Keer et al., is apoptosis [132]. However, a recurring problem with apoptosis is cancer cells’ resistance to this type of cell death when using anticancer drugs [133,134]. The increasing amount of research in medicinal chemistry has led to a detailed list of RCD mechanisms, especially in developing therapeutics for cancers and other diseases [135]. Previously, cell death mechanisms in mammals were classified as apoptosis, necroptosis, or autophagy-dependent; however, new forms of RCD have been discovered, including pyroptosis [136] and ferroptosis [56]. Ferroptosis was discovered when a group of scientists were developing therapeutic drugs targeting a mutated proto-oncogene involved in cancer, RAS [50,56]. These researchers were able to find two compounds toxic to cancer cells expressing mutated RAS, RSL3, and erastin [50,56]. The type of cell death followed by the treatment with these two drugs did not fit into any of the already discovered RCD mechanisms [50,56]. Since this cell death mechanism was shown to be ineffective in the presence of deferoxamine, an iron chelator, ferroptosis was then defined as an iron-dependent process that originates membrane damage through lipid peroxidation [47,50,56]. Ferroptosis has been linked with multiple diseases, since inducing this type of RCD seems to be a potential therapy for Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, lung cancer, prostate cancer, breast cancer, melanoma, hepatocellular carcinoma, pancreatic cancer, and skin cancer. Therefore, the therapeutic potential of ferroptosis in this field is immense, and its relation to other diseases is also emerging. Back in the 1950s, Harry Eagle was able to demonstrate how cell death was achieved by cysteine depletion and the consequent reduction of glutathione. In contrast, cysteine synthesis did not reduce glutathione but restored its levels, causing cell death cessation [137,138]. A few years after, a type of vitamin E, alpha-tocopherol, was identified by Shiro Bannai et al. as an antioxidant capable of reversing cell death, regardless of the glutathione levels and no cystine present [139]. Finally, in 2012, “ferroptosis” was distinguished from other RCD forms and was defined as glutamate-induced cell death [140]. Ferroptosis was characterized as sensitive to iron and lipid peroxidation due to its inefficacy in the presence of an iron chelator and an antioxidant, respectively. In general, ferroptosis results in morphological changes, including a reduced mitochondrial volume, increased density of the lipid bilayer membrane, disrupted outer mitochondrial membrane, dwindled mitochondrial cristae, swelled cells, and ruptured plasma membrane [141,142]. Due to the increased metabolic demand for iron to support cancer cell growth, ferroptosis can be used for therapeutic purposes. Therapeutic strategies can disrupt this metal homeostasis and therefore trigger ferroptosis, resulting in an inhibition of cell proliferation, cell death, and tumor growth. It is well known that tumors have an increased metabolic demand for iron to support their growth, and this could be turned to therapeutic purposes by deregulating the metal homeostasis and triggering this type of programmed cell death. Biochemically, ferroptosis leads to glutathione depletion, a subsequent decrease in GPX4 activity, mevalonate-pathway-derived coenzyme Q10 (CoQ₁₀) depletion, and consequent SQS activation [143,144,145]. Additionally, the mevalonate pathway final product, cholesterol, has been closely related to this programmed cell death type. Abundant cholesterol metabolites’ availability has shown to increase the capacity of tumor and metastatic cells by upregulating cellular uptake and lipid biosynthesis [146]. This relation is not surprising as cholesterol composes part of cell membranes and is extremely susceptible to oxidation, especially by hydroxyl radicals [146]. These findings foreshadow the role that SQS can potentially play in the regulation of ferroptosis and, therefore, in the therapeutic field across many diseases.

Ferroptosis is driven by an imbalance in the levels of oxidants and antioxidants that results in lipid peroxidation, a process by which free radicals or reactive oxygen species (ROS) attack lipids containing double-bounded carbons (C=C), especially polyunsaturated fatty acids (PUFAs) present in the cell membrane [143,147] Cells often resolve lipid damage through phospholipid peroxidase glutathione peroxidase 4 (GPX4) as an antioxidant-reducing toxic hydroperoxide [148]. Ferroptosis is characterized by the production and accumulation of lipid peroxides and the failure of internal mechanisms to eliminate them [149]. One potential explanation for this phenomenon involves the suppression of GPX4, thereby triggering a process known as ferroptosis cell death [147]. The resulting accumulation of lipid peroxides reaches lethal levels, which causes damage to the phospholipids conforming the lipid bilayer of the cell membrane, rupturing the cell and triggering ferroptosis cell death [150]. Three main regulatory levels of ferroptosis have been identified: (1) system Xc/reduced glutathione/glutathione peroxidase 4, (2) nicotinamide adenine dinucleotide phosphate/ferroptosis suppressor protein 1/coenzyme Q10 (CoQ10), and (3) guanosine triphosphate (GTP) cyclohydrolase 1/tetrahydrobiopterin/dihydrofolate reductase [149,151]. Regulatory systems (1) and (2) have been shown to be downregulated when SQS is active, as shown by the usage of class III ferroptosis inducers (FINs) [151,152]. For regulatory level (1), once SQS is activated through the binding of FIN56, GPX4 is depleted and inactivated, resulting in the rapid accumulation of ROS. Nevertheless, these lipid ROS can be repressed with iron chelators and lipophilic radical traps [152]. For regulatory level (2), the available FPP suffers competition between the different paths that it can take, including the formation of CoQ10 or the reaction on SQS to form squalene, based on the schematics of the mevalonate pathway [153].

Squalene is an oleophilic metabolite that accumulates in lipid droplets or cell membranes, especially in anaplastic large cell lymphoma (ALCL) cell lines. Hence, ferroptotic cell death and lipid peroxidation can potentially be regulated by SQS, similarly to GPX4 [44]. Cell lines not expressing SQLE, commonly ALCL, experience an accumulation of squalene due to the non-use of it in the cholesterol biosynthesis pathway (mevalonate pathway). SQLE is directly downstream from SQS in the mevalonate pathway. Through CRISP-9 experiments, SQS knockdown results in a decrease of squalene storage back to non-ALCL cell levels. Other experiments showed decreased tumor volume and size when knocking down FDFT1 due to removing squalene accumulation. Overall, these results suggest that SQS promotes optimal growth in ALCL cells through squalene accumulation [44]. Silencing FDTF1 and blocking squalene accumulation resulted in ferroptosis in ALCL cells when GPX4 was inhibited [44]. Supplementing the cells with extracellular squalene did not balance out FDFT1 knockdown, indicating that squalene must accumulate in the correct compartments, ER membranes, to protect against ferroptotic cell death [44]. These findings were further supported using an antioxidant (ferrostatin-1), which showed a decreased sensitivity to GPX4 inhibitors in FDFT1 knockdown ALCL cells [44]. Similarly, expression of SQLE in ALCL cells removing the squalene accumulation resulted in a decrease in tumor growth [44]. Removal of the squalene accumulation via FDFT1 knockdown, SQLE expression, or SQS inhibition has been associated with an overexpression of lipid ROS, a feature of ferroptosis [44]. FIN56, a small-molecule inducer of ferroptosis, has been tested on the human fibrosarcoma HT1080 cell line (these cells are from human epithelial tissue obtained from a fibrosarcoma patient) [154], and through short hairpin RNAs (shRNAs) against FDFT1. The data suggest that FIN56 activates SQS. Additionally, inhibiting SQS with known inhibitors (YM-53601 and zaragozic acid A) has shown a suppression in FIN56 lethality. The binding of hSQS protein to an affinity probe vanished by pre-incubation of purified SQS with FIN56, suggests a binding interaction between SQS and FIN56 [154]. It is also known that SQS’s substrate, FPP, participates in other processes besides cholesterol biosynthesis, such as synthesizing sterols, CoQ10, dolichol, ubiquinone, and heme A. Supplementation of FPP, SQS inhibition, and SQLE inhibition separately suppressed FIN56 lethality in HT1080 cells [154]. These results suggest the idea of certain metabolites derived from FPP regulating the cytotoxicity of FIN56. CoQ10 was the only one to suppress cell death via FIN56-induced ferroptosis among the four known metabolites. Thus, CoQ10 is thought to regulate FIN56 activity. However, CoQ10 supplementation has not been effective due to its extreme hydrophobicity and SQS being in a hydrophilic environment [50]. The combination of these results suggests that squalene can protect against chemical modifications when PUFAs membranes are damaged via oxidation. Loss of squalene accumulation in the ER membrane has also shown the depletion of PUFAs membranes. Although GPX4 is considered protective against lipid peroxidation, no change in GPX4 protein levels has been observed with FDFT1 knockdown. Researchers have concluded that squalene is an upstream metabolite in the cholesterol biosynthesis pathway that protects against peroxidation, whereas its absence promotes ferroptosis [44,47,50]. Additionally, CoQ10 is thought to regulate FIN56 lethality, but its supplementation as an effective technique to suppress ferroptosis is held back due to its extreme hydrophobicity. In other words, squalene is considered to possess anti-ferroptosis properties in certain cancer cells [44].

Squalene synthase has been launched into the research field as a potential therapeutic target because its product, squalene, protects cellular membranes from oxidative damage and participates in the mevalonate pathway. Its potential protective function against ferroptosis and lipid peroxidation is cognate to the accumulation of squalene in the endoplasmic reticulum membrane, which acts as an antioxidant and a lipophilic radical trap. Accordingly, SQS has been identified as a regulator of not only ferroptosis cell death pathways but also other diseases, ranging from viruses to cancers. In the past 20 years, an increasing amount of research has been conducted on SQS, which has been discovered in an extensive number of inhibitors and modulators. Even though its biochemical structure, reaction mechanism, significance in diseases, and relation to ferroptosis have been discovered, much remains to be learnt about the regulation and therapeutic usage of SQS. Increasing the knowledge on how SQS inhibition in humans could be achieved as an alternative assignment for other treatments such as chemotherapy, radiotherapy, and statins. Due to the challenges experienced during past clinical trials, there is a limitation in the current inhibitors. The continued development of new SQS inhibitors with improved drug-like and pharmacokinetic properties and their ultimate clinical trials may facilitate the pharmaceutical development of compounds targeting this enzyme. Therefore, the research field hopes that a better knowledge of SQS’s regulation and therapeutic usage can promote clinical development in treating cancers and other conditions.