The Contribution of Cholesterol and Squalene Synthase in Cancer: Molecular Mechanisms, Lipid Rafts and Therapeutic Approaches.

1
Introduction
Cholesterol, first identified in human gallstones in 1769 [1], is predominantly located in cell membranes [2], regulating its properties [3] and forming dynamic microdomains on the plasma membrane, called lipid rafts [4]. Beyond membrane functions, cholesterol generates various derivatives with significant physiological roles, such as oxysterols, bile acids, and steroid hormones [5]. While its connection to cardiovascular diseases is well established [6], emerging evidence links cholesterol to other pathological conditions, including neurodegenerative diseases [7] and cancer [8, 9]. Epidemiological studies indicate that there is an association between cancer and serum cholesterol levels; however, these results are controversial as they depend on cancer type, comorbidities, therapeutic interventions, and menopausal status [10, 11, 12, 13, 14]. The association between dietary cholesterol uptake and cancer risk is equally contradictory [11, 15]. Recent reviews by Huang et al. [5], Göbel et al. [16], Xu et al. [17], Liu et al. [18], and Giacomini et al. [19] refer to some aspects of the intricate interplay between cholesterol metabolism and both carcinogenesis and cancer progression. Ediriweera [20] discusses recent advances in anticancer therapeutic approaches centered around cholesterol metabolism. Further, Codini et al. [21] and Li et al. [22] attempt to elucidate the involvement of cholesterol in lipid raft formation in cancer.
In this comprehensive review, we explore the diverse mechanisms by which cholesterol metabolism is disrupted, the intricate mechanisms by which cholesterol and its metabolites participate in carcinogenesis and metastasis, and, in particular, the significant role of cholesterol in lipid raft formation as well as the mechanisms by which lipid rafts are involved in cancer. The above‐mentioned aspects are elaborated in view of the therapeutic potential of targeting SQS; squalene synthase (SQS; also called farnesyldiphosphate farnesyltransferase 1, FDFT1) is a pivotal enzyme in the mevalonate pathway, and through its selective inhibition, only sterol (i.e., cholesterol) synthesis is impeded [23]. The aim is to comprehend the anticancer effect of SQS inhibition by presenting the diverse mechanisms by which SQS is implicated in cancer and metastasis and by building upon the latest insights and research findings in the field.
1.1
Intracellular Cholesterol Homeostasis Dysregulation in Cancer
Cellular cholesterol levels are strictly regulated by biosynthesis, uptake, export, and storage (Figure 1). De novo biosynthesis takes place through the mevalonate pathway from acetyl‐CoA through approximately 30 reactions. It starts with two acetyl‐CoA molecules merging, followed by the addition of a third acetyl‐CoA, creating 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA). HMG‐CoA is then converted into mevalonic acid via the enzyme HMG‐CoA reductase (HMGCR), a primary rate‐limiting step in cholesterol synthesis and statins’ target. Through several stages, mevalonic acid produces isopentenyl‐pyrophosphate (IPP), which generates farnesyl‐pyrophosphate (FPP). SQS catalyzes the fusion of two FPP molecules to produce squalene, which is converted to squalene epoxide by squalene epoxidase (SQLE). Squalene epoxide undergoes cyclization and eventually transforms into cholesterol via intermediate steps. Alternatively, FPP can be directed to the non‐sterol branch of the pathway, where it generates geranylgeranyl‐pyrophosphate (GGPP). FPP and GGPP mediate posttranslational protein modification by protein prenylation. Moreover, the non‐sterol branch produces ubiquinone (Coenzyme Q10), dolichols, and heme A [24].
The most important regulator of the mevalonate pathway is the transcription factor called sterol regulatory element‐binding protein‐2 (SREBP2). Enterocytes or hepatocytes acquire cholesterol by uptake of low‐density lipoprotein (LDL) through LDL receptors (LDLR). Additionally, dietary cholesterol uptake is regulated by Niemann–Pick type C1‐like 1 (NPC1L1) protein. Excess cholesterol is exported via ATP‐binding cassette (ABC) transporters. Alternatively, cholesterol can be converted to cholesteryl ester by acyl‐CoA:cholesterol acyltransferase (ACAT; also called sterol O‐acyltransferase 1 (SOAT1)) for storage in lipid vesicles or secretion as lipoproteins [24]. Moreover, Liver X receptors (LXR; also known as oxysterol receptor LXR) play a crucial role in controlling overall cholesterol homeostasis [25]. For comprehensive details on cholesterol homeostasis mechanisms, refer to the review by Luo et al. [24]; our focus will be on dysregulated pathways in cancer.
Cancer cells show alterations in their metabolism to deal with the high energy and biosynthetic requirements due to rapid tumor growth [26]. Deregulation of enzymes and transcription factors in the mevalonate pathway and related signaling pathways plays an important role in malignancy development [27]. Overexpression and hyperactivity of mevalonate pathway enzymes appear in many cancers, and this phenomenon is associated with reduced recurrence‐free survival and overall clinical prognosis [16, 28, 29, 30, 31, 32, 33].
Cholesterol biosynthesis is a strictly controlled process that is highly dependent on SREBP transcription factors. The SREBP2 isoform is the main transcription factor regulating genes associated with the mevalonate pathway and is found in the endoplasmic reticulum, where it is bound to the SREBP‐cleavage activating protein (SCAP) (Figure 2). SCAP detects cholesterol levels in the endoplasmic reticulum, and upon sterol deficiency, the SREBP2‐SCAP complex dissociates from insulin‐induced genes (INSIGs) and moves to the Golgi apparatus. There, SREBP2 is converted by proteases to an active transcription factor (nSREBP2) that is eventually transported to the nucleus, binding to sterol regulatory element (SRE) promoter regions of various genes and finally, upregulating LDLR, HMGCR, SQS, and other mevalonate pathway enzymes. Cholesterol and its derivatives, such as oxysterols, inhibit the translocation of the SREBP‐SCAP complex, downregulating the SREBP2 pathway [24].
SREBP transcription factors are often dysregulated in various cancers, thus supporting cancer growth/metastasis, and are linked to a poor clinical profile [34, 35]. Elevated levels of SREBP in prostate cancer due to inhibition of certain microRNAs lead to carcinogenesis in vitro [36]. In cases where lipids and/or oxygen are limited, e.g., in the glioblastoma microenvironment, the transcription factor SREBP2 and its downstream targets, such as mevalonate pathway enzymes, are significantly stimulated [37]. In hepatocellular carcinoma, stabilized SREBP2 augments cholesterol biosynthesis, promotes tumorigenesis [38], and upregulates the expression of oncogenes, which contribute to tumor metastasis and growth [39]. In addition, androgen receptors in prostate cancer mediate increased SCAP expression, SREBP activation, and decreased cholesterol efflux [40]. In breast cancer, the increased activity of SREBP induces cell proliferation; its increased expression is associated with breast cancer cell metastasis and can also be a prognostic biomarker [35]. In endometrial cancer, SREBP expression is elevated, whereas SREBP inhibition leads to cancer cell apoptosis [41].

LXRs are nuclear receptors activated by endogenous oxysterols that play a central role in cholesterol homeostasis [42]. They regulate cholesterol elimination by promoting efflux, reducing cellular uptake, enhancing conversion to bile acids, and inhibiting de novo cholesterol synthesis [25]. These regulatory functions are achieved through several mechanisms: LXRs induce the expression of ABC transporters, facilitating cholesterol excretion into bile or the intestinal lumen. They also promote the degradation of LDLRs via the E3 ubiquitin ligase known as the inducible degrader of LDLR (IDOL), thereby decreasing LDL cholesterol uptake. In addition, LXRs downregulate the expression of NPC1L1, reducing intestinal cholesterol absorption, and activate CYP7A1, the rate‐limiting enzyme in bile acid synthesis, further promoting sterol excretion. Furthermore, LXRs redirect acetyl‐CoA from cholesterol biosynthesis towards fatty acid synthesis by upregulating lipogenic genes, including SREBP‐1c, fatty acid synthase (FAS), and stearoyl‐CoA desaturase (SCD1) [43].
In cancer, the relationship between intracellular cholesterol levels and LXR activation is often disrupted. LXRs play complex roles in oncogenesis by regulating numerous genes and pathways [44]. These include cholesterol‐dependent mechanisms and different cancer pathways, such as inhibition of Wnt signaling, PI3/Akt pathways, and modulation of antitumor immunity in the TME [45]. Differential LXR expression across cancers further underscores their significance. Reduced LXR expression correlates with poor prognosis in breast [46], prostate [47], liver [48], and colorectal cancer [49]. In hepatocellular carcinomas, for instance, c‐FOS—a critical regulator of tumorigenesis—leads to decreased LXR expression and activity, resulting in cholesterol accumulation and inflammation [50].

Phosphatidylinositol‐3‐kinase (PI3K)/Akt and mechanistic target of rapamycin (mTOR) are closely linked signaling pathways that are vital for cell growth and survival under both normal and pathological conditions; they are hereby discussed in relation to their regulation of the SREBP transcription factor, and indirectly, to cholesterol biosynthesis. The PI3K/Akt pathway, frequently altered in cancer, is essential for cell survival and proliferation in response to growth factors. Common occurrences in cancer include inactivating mutations in the tumor suppressor phosphatase and tensin homolog (PTEN) and hyperactivity of growth factor signaling, leading to enhanced signaling and increased cancer cell proliferation [51, 52]. mTOR, a serine/threonine kinase present in all mammalian cells, regulates signals from nutrient intake and growth factors, influencing protein synthesis and cell growth. The PI3K/Akt and mTOR complex 1 (mTORC1) signaling pathway exhibits increased activity in various cancers and plays an important role in cell growth and proliferation [53]. The role of the PI3K/Akt/mTORC1 signaling pathway in cancer progression has been extensively studied. For example, in prostate cancer, Akt‐induced increase in intracellular cholesterol levels leads to increased aggression [54] and tendency to metastasize to bones [55].
Akt is a serine/threonine protein kinase that regulates/enhances various cellular functions, such as metabolism, chemotaxis, cell cycle, migration, angiogenesis, apoptotic suppression, and cell survival. Akt is dysregulated in different types of cancer as follows (Figure 3): due to the inactivation of the tumor suppressor PTEN, the dephosphorylation of 3,4,5‐phosphatidyl‐inositol triphosphate (PIP3) is inhibited, prolonging its action and activating the PI3K/Akt pathway [56]. Akt induces the action of the transcription factor SREBP by enhancing its trafficking, by inhibiting its degradation, and by phosphorylating its precursor [57, 58]. mTORC1, a serine/threonine kinase, is a major downstream effector of the PI3K/Akt signaling pathway, which activates transcription factors essential for cell survival, including the transcription factor SREBP. mTORC1 is involved in the regulation of SREBP by inhibiting the entry of lipin‐1 phosphatidic acid phosphatase into the nucleus, and as a result, nuclear SREBP levels are elevated [59]. Furthermore, mTORC1 induces SREBP transport from the endoplasmic reticulum to the Golgi apparatus [60]. The enhancement of the PI3K/Akt/mTORC1‐SREBP axis increases the expression of genes associated with cholesterol and fatty acid synthesis, which consequently stimulates cancer cell proliferation and tumorigenesis [61, 62].
The transcription factor hypoxia‐inducible factor 1‐alpha (HIF1A) is the major regulator of oxygen homeostasis and regulates cell adaptation to hypoxic conditions [63]. Due to the rapid growth of cancer cells without the required blood supply, the tumor microenvironment (TME) is hypoxic [64]. Because of this hypoxia and several mutations, including that of p53, HIF1A is induced [65]. Accumulation of HIF1A leads to induced transcription and increased activity of HMGCR [66]. Thus, activation of the transcription factor HIF1A under carcinogenic conditions further induces the growth of cancer cells by stimulating the mevalonate pathway.
Additionally, the TME is acidic, i.e., pH 6.8, compared to 7.4 normally, due to glycolysis that leads to the secretion of protons and lactic acid. These conditions favor the transfer of SREBP2 to the nucleus and its binding to the promoter of target genes. HMG‐CoA synthase, SQS, and SQLE are influenced the most, resulting in increased cholesterol biosynthesis. Activation of SREBP2 under acidic conditions has been observed in pancreatic cancer, glioma, renal carcinoma, sarcoma, invasive breast carcinoma, esophageal or thyroid carcinoma, and rectum adenocarcinoma [67].
Furthermore, inhibition of ACAT1, and therefore inhibition of cholesterol esterification, has been found to reduce tumor growth in various cancers, including prostate, pancreatic, and liver cancer, through different suggested mechanisms. ACAT inhibition is associated with apoptosis by free cholesterol accumulation in the endoplasmic reticulum. The endoplasmic reticulum, inherently low in cholesterol, is vulnerable to elevated levels of cholesterol, triggering an endoplasmic reticulum stress pathway [68], and a new strategy for treating metastatic pancreatic cancer is based on this mechanism [69]. Moreover, the S‐III subtype of hepatocellular carcinoma, characterized by disrupted cholesterol homeostasis and elevated ACAT1 expression, exhibits altered cholesterol distribution upon ACAT1 knockdown. This alteration reduces plasma membrane cholesterol content, suppressing cancer proliferation and migration by inhibiting tumorigenic signaling pathways [70]. In prostate cancer, inhibiting ACAT leads to SREBP and LDLR downregulation through a negative feedback loop triggered by elevated free cholesterol levels, and thus leads to a reduction of de novo synthesis and uptake of cholesterol. Prostate cancer cells use the esterified form of cholesterol to avoid excess cholesterol toxicity and maintain active SREBP. Depleting cholesterol ester storage disrupts intracellular cholesterol homeostasis, reducing essential fatty acid uptake and potentially inducing apoptosis due to elevated free cholesterol levels. The altered cholesterol metabolism in prostate cancer may also affect lipid raft regulation [54].
ABC transporters, situated in cell membranes and intracellular organelle membranes, are responsible for the efflux of various substrates. The ABC transporter A1 (ABCA1) exports cholesterol from the cell to form HDL particles, and also facilitates cholesterol transport from the cellular membrane to the endoplasmic reticulum [71]. Epidemiological and experimental findings suggest that ABCA1 has anticancer activity in tumor development; however, downregulation of ABCA1 is also linked to carcinogenesis. Notably, further research is needed to fully understand its role [72].
In most cancers, the tumor suppressor p53 is not functional due to mutations. Under normal conditions, p53 is a crucial tumor suppressor; its various roles include cell cycle disruption, cell death, senescence, and DNA repair, while also regulating metabolic pathways to inhibit cell growth [73, 74]. One of the major pathways regulated by p53 is the mevalonate pathway by inhibiting the expression of genes involved in it. The effect of p53 on the mevalonate pathway is responsible for its tumor suppressor effect in liver cancer. More specifically, the p53 protein induces the activation of ABCA1, which transports cholesterol to the endoplasmic reticulum, thereby inhibiting SREBP2 maturation and the mevalonate pathway. However, when the p53 tumor suppressor protein is mutated, which is common in hepatocellular carcinoma, the expression of ABCA1 is reduced, the action of the transcription factor SREBP2 is induced, and the mevalonate pathway re‐operates to supply the needs of cancer cells. Hepatocellular carcinoma has no effective treatment, and therefore, mevalonate pathway inhibitors are very promising in addressing the lack of tumor suppressor protein p53 [75].
Recent studies underscore the critical role of the MYC oncogene in disrupting cholesterol homeostasis in cancer. MYC, a frequently overexpressed transcription factor in lung cancer, causes deregulation of cholesterol uptake and efflux. Its activation leads to intracellular cholesterol accumulation, which is converted to cholesteryl esters and stored in lipid droplets; this mechanism allows cancer cells to manage and reserve excess cholesterol. MYC regulates cholesterol metabolism primarily by directly binding to and upregulating SQLE and thus inducing cholesterol biosynthesis. Additionally, cholesterol accumulation associated with MYC activation is suggested to be linked to LXR deactivation and SREBP induction [76, 77, 78].
Another well‐known cancer pathway that also affects cholesterol homeostasis is the nuclear factor‐κB (NF‐κB). NF‐κB exhibits pleiotropic, context‐dependent effects, acting as both a tumor promoter and, in some cases, a tumor suppressor [79]. In liver cancer cells, activation of NF‐κB pro‐inflammatory signaling upregulates key enzymes involved in cholesterol uptake and biosynthesis, including HMGCR, LDLR, and SREBP2, leading to cholesterol accumulation, which further promotes NF‐κB signaling [80]. On the contrary, in macrophages, cholesterol accumulation by ABCG1 loss increases NF‐κB activity, promoting in this way a tumor‐fighting phenotype [81].
The dysregulation of cellular cholesterol homeostasis is intricately linked to cancer development, influencing crucial signaling pathways and metabolic processes. Disruptions in the mevalonate pathway, orchestrated by transcription factors such as SREBP2, contribute to the aggressive behavior of malignancies. Well‐established cancer‐related signaling pathways, including PI3K/Akt/mTORC1, p53, MYC, NF‐κB, and HIF1A, intersect with cholesterol metabolism to support cancer progression and survival. Emerging evidence highlights the role of cholesterol dysregulation in fostering therapy resistance and supporting oncogenic pathways across diverse cancer models. These insights underscore the potential of targeting cholesterol homeostasis mechanisms, including the inhibition of ACAT1 or modulation of the mevalonate pathway, to disrupt tumor growth and enhance treatment outcomes. Thus, the intricate interplay between cholesterol homeostasis and oncogenic mechanisms unveils a spectrum of potential targets for the development of innovative cancer therapies, positioning cholesterol metabolism as a promising focal point in oncology research.

1.2
The Role of Cholesterol in the Development and Progression of Cancer
In general, cholesterol plays an important role in cancer development. Elevated cholesterol levels are associated with a higher occurrence of some cancer types, such as breast and prostate cancer. Administration of drugs that reduce cholesterol levels shows favorable results by reducing the risk and mortality. Intracellular accumulation of cholesterol in malignancies is well established; increased uptake as well as reduced efflux occurs to meet the increased requirements for membrane biosynthesis and other functions [82].
Among others, cells need cholesterol to proliferate; once entering the process of division, they induce the mevalonate pathway and LDLR expression to fulfill the adequate amounts of cholesterol. Thus, rapidly proliferating cells such as cancer cells exhibit accelerated cholesterol biosynthesis. Cholesterol is necessary in various stages of cell cycle progression. By inhibiting the biosynthesis of cholesterol, cancer cell division is also inhibited. Cholesterol synthesis is increased during the G1 phase, where the cell prepares for division, as well as during the G2/M phase, mainly due to the phosphorylation of the SREBP1 transcription factor. Inhibition of cholesterol synthesis in vitro by statins or other drug molecules resulted in termination of the cell cycle at the G1 phase due to inactivation of various cyclin‐dependent kinases, the main cell cycle regulators. In particular, mevalonate and its non‐sterol derivatives are required for the transition from phase G1 to phase S, while cholesterol is needed to complete mitosis. For example, the administration of SQS inhibitors leads to cell accumulation in the G2/M phase (Figure 4) [83].
Cholesterol is also involved in the Hedgehog signaling pathway. This signaling pathway plays a key role in embryogenesis and subsequent tissue regeneration. Hedgehog proteins control cell growth, survival, and differentiation through different mechanisms according to the cell type. As in other pathways responsible for growth, a disturbance in its activity can lead to carcinogenesis, e.g., basal cell carcinoma. This signaling pathway is involved in the development of cancer primarily through inhibition of apoptosis and induction of angiogenesis. Two receptors are mainly involved in the Hedgehog pathway, the Patched receptor and the Smoothened receptor; the former inhibits the activation of the latter. Cholesterol binds directly and covalently to the Smoothened receptor and activates it, thereby inducing its signal transduction pathway [84, 85].
In addition, cholesterol appears to enhance cancer resistance to chemotherapy. For example, in colon cancer resistant to destruxins (secondary fungal metabolites with anticancer activity), the biosynthesis of cholesterol is significantly increased. As a result, the cytoplasmic membrane of cancer cells is rearranged, and the ionophore activity of these drugs is reduced. Inhibition of the mevalonate pathway significantly reduces resistance to destruxins, while cholesterol replenishment protects cells that are sensitive to destruxins from their cytotoxic effects [86].
Cholesterol and changes in the lipid composition of membranes can alter the expression and activity of ABC transporter proteins, which also serve as multidrug efflux pumps, providing cancer cells with multidrug resistance (MDR). One explanation is that cells with MDR phenotype have altered their cell membrane composition through enrichment with long‐chain saturated lipids and cholesterol, thereby reducing fluidity and enhancing the presence of so‐called lipid rafts. This change affects the conformation of the transmembrane region of ABC transporter proteins, favoring the binding and outward release of drug molecules. Also, along with the fluidity, the permeability of the membrane is disturbed, thus preventing the passive diffusion of hydrophilic or amphiphilic drug molecules. Another explanation is that MDR cancer cells have modified the composition of organelle membranes (mitochondria and endoplasmic reticulum) to contain fewer fatty acids susceptible to oxidation. Therefore, these cells are more resistant to oxidative stress and apoptosis caused by chemotherapy [87].

Steroid hormones regulate cell proliferation and differentiation and are directly linked to prostate cancer and breast cancer. Androgen deprivation is a common treatment for prostate cancer, as is the inhibition of estrogen‐synthesizing aromatases for breast cancer. However, an abundance of cholesterol, as a precursor to steroid hormones, allows for a continuous intracellular production of androgens and estrogens in hormone‐sensitive prostate and breast cancers, which in turn activate androgen and estrogen receptors, respectively. Thus, cholesterol makes hormone‐sensitive cancer cells of prostate and breast cancers independent of circulating hormones, making castration or aromatase inhibitors, respectively, ineffective [88, 89, 90, 91]. In hormone‐sensitive breast cancer, oxysterols, which are oxygenated derivatives of cholesterol, play a dominant role. 27‐Hydroxycholesterol binds to estrogen receptors, thereby inducing the development of breast cancer. When long‐term reduced estrogen levels occur in breast cancer cells due to treatment with aromatase inhibitors, oxysterols replace estrogens and activate estrogen receptors, resulting in resistance development [92, 93].
However, oxysterols play a complex role in carcinogenesis. On the one hand, they promote cancer growth and metastasis; for instance, in breast cancer, oxysterols support proliferation, invasion, and induce hormonal therapy resistance through binding to the estrogen receptor. In prostate cancer, oxysterols promote proliferation via androgen receptor activation. Elevated oxysterol levels are associated not only with breast and prostate cancer but also with colon, lung, skin, bile duct cancer, and cholangiocarcinoma [94, 95]. On the other hand, oxysterols exhibit antiproliferative characteristics. 24‐, 25‐, and 27‐hydroxycholesterol are the main endogenous activators of the LXR, thus eliminating cholesterol by promoting efflux, reducing cellular uptake, enhancing conversion to bile acids, and inhibiting de novo synthesis. Additionally, oxysterols bind to INSIG, stabilizing the SREBP‐SCAP complex in the endoplasmatic reticulum and inhibiting SREBP through an LXR‐independent mechanism [43].
Cholesterol and oxysterols are essential in cells of the immune system, where they regulate inflammatory reactions and innate immunity [96]. Recently, it has been shown that cholesterol and oxysterols are associated with CD8+ tumor‐infiltrating lymphocytes (CD8+TILs) with controversial, however, effects; they can either enhance their function or lead to their exhaustion and dysfunction [97]. In the TME, prevalent hypoxia and lack of nutrients lead to endoplasmic reticulum stress, i.e., a condition where unfolded or incorrectly folded proteins accumulate in the endoplasmic reticulum. In lung cancer with excess cholesterol or oxysterol, CD8+TILs absorb excess cholesterol in the TME, resulting in endoplasmic reticulum stress and eventual exhaustion of CD8+TILs [98]. However, another study showed that the accumulation of cholesterol in the plasma membrane of CD8+TILs, due to inhibition of ACAT1, facilitates T‐cell receptors to form clusters and immunological synapses, thereby increasing their anticancer action. Especially in melanoma, inhibition of ACAT1 enhances the effect of immunotherapy [99]. Consequently, the role of cholesterol in various tumor‐infiltrating cells in different cancers would be interesting to clarify. Oxysterols hinder the action of dendritic cells, which are antigen‐presenting cells. By activating LXRs, they inhibit the expression of the CC chemokine receptor type 7 on the surface of dendritic cells, thereby suppressing their migration to lymphoid organs. As a result, oxysterols inhibit cancer antigen presentation to T and B cells and finally inhibit T‐cell anticancer activity [100]. However, another study supports that cholesterol efflux from tumor‐associated macrophages (TAMs) induced by ovarian cancer cells causes their phenotype transition from tumor‐suppressing to tumor‐supportive [101].
Excess cholesterol in the endoplasmatic reticulum is sensed by the Nuclear Factor Erythroid 2‐Related Factor 1 (NRF1). NRF1 plays a critical role in maintaining lipid and cholesterol homeostasis through directly binding to cholesterol, enabling the endoplasmatic reticulum to detect and counteract excess cholesterol, thereby preventing hepatic cholesterol accumulation and damage. It achieves this by suppressing CD36‐driven inflammatory signaling, activating LXR activity, and facilitating cholesterol excretion [102, 103, 104]. Pharmacological strategies aimed at restoring NRF1 activity may offer novel approaches to mitigate the cytotoxic effects of cholesterol accumulation in cancer and other diseases.
It should be noted that the outcomes of clinical studies investigating the connection between cholesterol levels and carcinogenesis are sometimes contradictory. However, preclinical research has more consistently indicated that imbalances in cholesterol homeostasis play a significant role in cancer onset [12].

1.3
The Role of Cholesterol and Its Metabolites in Cancer Metastasis
Metastasis is a pivotal phase in cancer progression. More than 90% of cancer mortality is associated with the development of a second tumor, i.e., metastasis to another organ [105]. Recent studies have highlighted the profound impact of cholesterol metabolites, especially 27‐hydroxycholesterol, on the interaction between cancer cells and the TME, which facilitates cancer metastasis, invasion, and angiogenesis.
The metastatic phenotype of cancer cells is driven by signaling pathways that reduce cell adhesion and enhance cell migration [106]. Tumor metastasis begins with a change in the interaction of cancer cells with the so‐called basement membrane, a special form of extracellular matrix [107]. The TME is formed by the extracellular matrix, cells such as fibroblasts, immune and endothelial cells, nerves, and blood vessels, and is essential for the onset, growth, and tumor treatment resistance. The relationship between cancer cells and their surrounding microenvironment has been extensively researched. Certain extracellular matrix elements, including specific collagens and matrix metalloproteinases (MMPs), drive invasion and metastasis by encouraging epithelial‐mesenchymal transition (EMT) [108]. During EMT, epithelial cells lose their typical structures, undergo cytoskeletal changes, and alter gene expression, enhancing cell movement and invasiveness, a process that is a prerequisite for the onset of metastasis [109]. Similarly, endothelial‐to‐mesenchymal transition (EndMT) involves the transformation of endothelial cells to mesenchymal cells, influencing tissue fibrosis and cancer [110]. The vascular system in the TME, through endothelial alteration, plays a key role in carcinogenesis and progression. Recent findings show that these impaired endothelial cells increase the levels of vascular endothelial growth factor (VEGF), further fueling blood vessel formation, cellular migration, and EMT [111]
Lipid profiles differ between epithelial and mesenchymal cells, and when cells transition through the EMT process, there is a noticeable rise in cholesterol content [112]. Cancer cells in the midst of EMT are more responsive to cholesterol‐reducing drugs such as statins, making mesenchymal cell plasma membrane less fluid and potentially diminishing cell movement and metastasis [113, 114]. Oxysterols can influence cellular functions in the TME, and 27‐hydroxycholesterol has a significant role in the metastasis process. 27‐Hydroxycholesterol induces invasion and migration of breast cancer cells by activation of the signal transducer and activator of transcription 3 (STAT3), which results in significantly increased expression of MMP‐9, which breaks down most extracellular matrix components, and finally in EMT promotion [115]. In a similar way, through STAT3 activation, 27‐hydroxycholesterol promotes EndMT in vascular endothelial cells, which further induces EMT, activation of MMP‐9, and consequently breast cancer cell migration [116]. In addition, inhibition of cholesterol biosynthesis can reduce VEGF levels by inhibiting VEGFR2 receptor dimerization in the endothelial cell membrane, thereby inhibiting endothelial migration and consequently inhibiting angiogenesis signaling [117]. Thus, cholesterol and 27‐hydroxycholesterol promote cellular transitions and metastatic processes as well as angiogenesis.
In breast cancer, 27‐hydroxycholesterol increases the levels of polymorphonuclear neutrophils and γδ T‐cells (cells with T‐cell receptors), thereby promoting angiogenesis and metastasis. It also decreases the levels of CD8 + T‐cells in distant metastatic sites, thereby inducing metastasis [118]. Furthermore, 27‐hydroxycholesterol induces the differentiation of osteoclasts by activating the STAT3 signaling pathway, as described previously, thus providing the appropriate microenvironment in bone tissue to be infiltrated by lung adenocarcinoma cells [119].
Finally, in response to 27‐hydroxycholesterol, cells increase lipid uptake and/or synthesis. The metabolic stress caused by lipid accumulation is counteracted by lipid glutathione peroxidase 4 (GPX4), which inhibits cell death caused by ferroptosis. Ferroptosis is a type of programmed iron‐dependent cell death characterized by the accumulation of lipid peroxides. Resistance to ferroptosis is a trait of metastatic cells; GPX4 elimination reduces the intense oncogenic and metastatic activity that cells acquire after long‐term exposure to 27‐hydroxycholesterol, a phenomenon found in patients with hypercholesterolemia. Cancers derived from mesenchymal cells or cancer cells that exhibit mesenchymal cell characteristics are particularly sensitive to ferroptosis and are therefore sensitive to drugs that inhibit GPX4 activity or expression [120].
In conclusion, cholesterol and especially cholesterol‐derived metabolites contribute substantially to cancer progression, metastasis, and invasion. Lowering cholesterol levels has shown the potential to prevent tumor growth and invasion in various types of cancer in preclinical and clinical studies [5].

1.4
The Role of Cholesterol in Lipid Rafts and Their Involvement in Cancer and Metastasis
The intricate relationship between cholesterol and cancer extends beyond its role in metabolism and cellular signaling, delving into its critical function within the plasma membrane. The role of cholesterol in the plasma membrane is diverse, as it is not limited to the regulation of fluidity and permeability but is also required for creating lipid rafts—sterol‐enriched membrane microdomains integral to cellular signaling and compartmentalization. In healthy cells, these domains work positively in various cellular processes, enhancing the communication of the cell with its microenvironment to produce and regulate biological responses. However, they can also serve as hubs for oncogenic signaling, fostering processes such as tumor cell adhesion, migration, and invasion. The present chapter explores the specific contributions of cholesterol to the structural and functional dynamics of lipid rafts in cancer. By examining the unique localization and behavior of key proteins within lipid rafts and their dependence on cholesterol, we highlight emerging insights into the molecular basis of cancer and present innovative therapeutic strategies that target these cholesterol‐dependent platforms [121].
Lipid rafts are small (10–200 nm), defined, heterogenous, sterol‐ and sphingolipid‐enriched membrane domains, characterized by a dynamic and transient nature. They contain saturated phospholipids, glycolipids, lipidated proteins, and glycosylphosphatidylinositol (GPI)‐anchored proteins (Figure 5). These microdomains compartmentalize various cellular activities, segregating specific elements to finely regulate their interactions with other membrane components and, consequently, regulate their functionality. The localization of a protein in a lipid raft can influence its conformation, thereby impacting its activity [4, 122]. Their roles include facilitating extracellular ligand‐receptor binding, initiating intracellular signaling pathways, modulating synaptic transmission, membrane trafficking, cytoskeleton organization, differentiation, apoptosis, cell adhesion to the extracellular matrix, and migration [21, 123, 124].
To support their rapid growth and aggressive progression, cancer cells enhance lipid metabolism and its associated signaling to meet the demands for membrane formation, energy storage, and the production of signaling molecules. The endogenous biosynthesis of lipids results in oncogenic stimuli that drive malignant tumor progression [121]. Compared to normal cells, cancer cells exhibit elevated levels of intracellular cholesterol and lipid rafts. A proteomic analysis of lipid rafts showed elevated interaction with the cytoskeleton and stronger protein‐protein interactions in breast, kidney, and melanoma cancer cells compared to normal cells, while a second analysis showed that lipid rafts are more stable in cancer cells than in normal cells [125]. Lipid rafts are closely associated with cancer‐supporting growth factor receptors and membrane adhesion mechanisms, serving as platforms for oncogenic signaling [126, 127]. Due to this high dependence on lipid rafts, cancer cells are more sensitive to cholesterol‐lowering agents than normal cells [128].
An outline of lipid raft involvement in cancer is shown in Figure 6; more specifically, lipid rafts are involved in various signaling pathways associated with cancer progression [22, 129]; they recruit and activate proteins, such as Akt kinase [130], Ras GTPase [131], Src kinase [132], c‐Met kinase [133], and induce oncogenic signaling. Wnt/β‐catenin [134] and Hedgehog [135, 136] pathways are well‐known cancer‐associated pathways that use lipid rafts as signaling platforms, whereas markers associated with cancer stem cells, such as CD44 glycoprotein [137] and CXCR4 chemokine receptors [138, 139], are also located within lipid rafts [140].
For example, the activation of protein kinase Akt when it is embedded in lipid rafts is more efficient. By adding methyl‐β‐cyclodextrin (MβCD), a cholesterol‐removing agent mainly used for lipid raft disruption, Akt phosphorylation becomes inhibited and its activation is hindered, ultimately inducing cancer cell apoptosis [130].
Ras proteins are GTPases with oncogenic action [141, 142]. The distinct localization patterns—within lipid rafts, lipid‐disordered domains, or at the lipid raft border—of activated or inactivated Ras isomers, including H‐, N‐, and K‐Ras, in specific cancer types provide valuable insights into the functional differences among these homologs. This advanced insight offers potential strategies for targeting these traditionally undruggable proteins by inhibiting their interactions with the cell membrane [143, 144].
c‐Met kinase is located in lipid rafts and supports cancer metastasis and resistance to radiotherapy. By disrupting lipid rafts through MβCD, c‐Met activation is inhibited, thereby reducing cancer cell proliferation and resistance to radiotherapy in non‐small cell lung cancer [133].
Lipid rafts accommodate growth factor receptors such as human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), the signaling of which depends on the concentration of cholesterol in the rafts. Reduction of cholesterol levels and therefore disruption of lipid rafts interferes with the activation of these receptors and causes inhibition of cell growth [145, 146]. Lipid rafts also play a significant role in angiogenesis signaling, as their disruption can lead to attenuation of angiogenesis via inhibiting endothelial cell migration and capillary formation [147], or angiogenesis signaling inhibition by suppressing tyrosine kinase receptors, which serve as proangiogenic markers [148]. In addition, VEGFR2, similarly to other growth factor receptors, is located in lipid rafts. Through LXR activation, endothelial cholesterol levels decrease, which in turn disrupts VEGFR2 localization within lipid rafts, leading to suppressed angiogenesis signaling [149].
The CXCL12 chemokine signaling pathway, with its CXCR4 receptor, induces metastasis in various types of cancer, particularly driving prostate cancer metastasis to bone. A crucial point is that the effectiveness of the CXCL12/CXCR4 pathway relies on its localization within lipid rafts, where it activates growth factors EGFR and HER2 and induces intraosseous tumor growth. Inhibition of this pathway appeared to hinder the onset of tumor growth but did not impact the growth of pre‐existing bone tumors, whereas inhibition of EGFR signaling led to growth suppression of established bone tumors [150]. In detail, binding of the chemokine CXCL12 to its receptor leads to a signaling cascade that activates MMP‐9; binding of CXCL12 to CXCR4 in lipid rafts induces the expression and release of PI3K and mitogen‐activated protein kinase (MAPK). Through their signaling pathways, Akt is activated, which in turn activates the transcription factor NF‐κB, resulting in the promotion of the expression of various genes, including that of MMP‐9 [151]. MMPs, as mentioned previously, are proteolytic enzymes that degrade the extracellular matrix. They are localized in lipid rafts, and their increased activity is associated with cancer infiltration and metastasis. Administration of MβCD inhibits the migration and invasion of breast cancer cells, which can also be attributed to the reduction of MMP‐9 in lipid rafts [152].
Many processes involved in metastasis, such as cell adhesion, migration, and EMT, are regulated by lipid rafts [153]. For example, cyclodextrin administration, which disrupts lipid rafts, reverses EMT in breast cancer [154], while nystatin administration, a cholesterol‐sequestering agent that also disrupts lipid rafts, reverses EMT in stomach cancer [155]. Many studies indicate that lipid rafts contain proteins necessary for EMT, such as CD44, caveolin‐1, and flotillins [21]. Elevated levels of caveolin‐1 are linked to EMT, heightened mobility in bladder cancer cell lines, and metastatic bladder cancer by activating the PI3K/Akt pathway, while caveolin‐1 knockdown inhibits EMT [156]. In addition, flotillins are upregulated in multiple cancer types and induce metastasis in diverse tumors, while they are associated with poor prognosis and lymph node metastasis, marking them as aggression indicators [157, 158].
Lipid rafts support adhesion and invasion of cancer cells. Many invasive cancer cells develop invadopodia—protrusive structures which contribute to extravasation, invasion, and extracellular matrix degradation [159]. In breast cancer cells, the formation of invadopodia and their involvement in extracellular matrix degradation rely on the presence of lipid rafts, which recruit MMPs on the surface of these protrusive structures [160, 161, 162].
Integrins are transmembrane receptors involved in intercellular interactions and cell‐extracellular matrix interactions. They bind to extracellular factors such as fibronectin, laminin, and other adhesion glycoproteins. When integrins attach to the extracellular matrix, intracellular signals are transmitted that control cell survival, proliferation, differentiation, migration, and cancer metastasis. Lipid rafts recruit activated integrins that interact with both upstream and downstream signaling molecules. Integrin localization within lipid rafts offers a more suitable membrane microenvironment that better accommodates their unique active conformation [163].
CD44 is a cell surface transmembrane glycoprotein that acts as a hyaluronan receptor and plays a pivotal role in cell adhesion, migration, and metastasis. It serves as an adhesion molecule that binds to lipid rafts and is expressed in various cancers [164], while cholesterol depletion disrupts its membrane localization and causes CD44 shedding, thereby reducing cancer cell adhesion and migration [165, 166]. Hyaluronic acid, which is prevalent in the extracellular matrix, is the primary ligand for CD44. This binding affinity is significantly influenced by CD44 localization within lipid rafts. High molecular weight hyaluronic acid molecules cause EMT through their binding with CD44, thus promoting metastasis [167]. Based on this binding mechanism, a nanosystem comprising MβCD and hyaluronic acid ceramide has been developed. This system targets and binds to CD44 receptor‐positive cancer cell surfaces via the hyaluronic acid moiety, while it disrupts lipid rafts via MβCD and is currently being explored for both cancer diagnosis and treatment [168].
In conclusion, advancing our understanding of lipid metabolism and its intricate roles in cancer cells will enable the identification of novel therapeutic targets. This knowledge holds the potential to significantly improve interventions aimed at inhibiting tumor progression and preventing metastasis.

1.5
Targeting Cholesterol Metabolism for Cancer Therapy
The mevalonate pathway is a potential target for cancer treatment, and statins, as HMGCR inhibitors, are therefore a useful tool. Their efficacy has been observed in preclinical in vitro and in vivo studies in various cancer types, including prostate, breast, colorectal, pancreatic, ovarian, lung, and cervical cancer, as well as hepatocellular carcinoma, leukemia, and myeloma [169]. Administering statins to cancer patients has shown an association with improved overall survival and reduced cancer‐related mortality [170, 171, 172, 173], although establishing a direct link between statin use and cancer risk reduction remains challenging [174].
Statins exert their anticancer effects through various mechanisms, both cholesterol‐dependent [175, 176, 177], through various mechanisms discussed previously, and cholesterol‐independent. These mechanisms include apoptosis activation, autophagy modulation, TME alteration, cellular plasticity reduction, angiogenesis inhibition, and suppression of cell cycle, proliferation, and invasion. These effects collectively lead to tumor growth suppression, reduced metastasis, and enhanced response to cancer therapy. The anticancer activity of statins is mainly based on the reduction of cholesterol levels, inhibition of isoprenoid synthesis, and production of reactive oxygen species (ROS). Additionally, statins influence key signaling pathways involved in carcinogenesis, such as p53, Akt, mTOR, VEGF, HIF1a, and chemokines [178, 179, 180].
Understanding the intricacies of statin‐induced anticancer effects proves challenging due to their diverse and complex actions. Previous studies suggested that statin‐induced effects on cancer and metastasis involve the inhibition of protein prenylation [181, 182, 183]. Statins lower FPP and GGPP levels, thereby inhibiting protein farnesylation and geranylgeranylation, impacting the function and localization of small GTPases like Ras and Rho, critical in cancer progression [175, 184, 185, 186, 187, 188, 189, 190]. For instance, GGPP increases the membrane localization of Rho GTPases, which in turn activate transcription cofactors and proto‐oncogenes YAP and TAZ, members of the Hippo signaling pathway, and finally promote carcinogenesis [191, 192]. However, another study has even revealed that statin‐induced cell death may be independent of Ras prenylation [193].
In cancer cells, ROS production is slightly elevated compared to normal cells, and to keep ROS within tolerable levels, cancer cells amplify their antioxidant defense systems. External oxidative stress exceeds their redox balance threshold and renders cancer cells more sensitive [194, 195]. Statins increase intracellular levels of ROS by inhibiting biosynthesis of isoprenoid antioxidants such as ubiquinone and dolichol; therefore, they exert some of their cytotoxic effects on cancer cells by increasing oxidative stress. For example, via this mechanism, statins cause apoptosis of lymphoma cells [196] and p53‐deficient colon cancer cells [197]. In addition, statins improve the sensitivity to chemotherapy due to oxidative stress induction [198, 199]. Simvastatin, through dolichol levels reduction, inhibits insulin‐like growth factor‐1 receptor (IGF1R) signaling and eventually reduces PC3 prostate cancer cell proliferation [200].
However, it is possible for cancer cells to develop statin resistance through alternative splicing of HMGCR [201] or by stimulation of the SREBP/HMGCR/LDLR feedback loop due to decreased cholesterol circulation. Upregulation of HMGCR in tumors during statin treatment raises concerns about a potential statin rebound effect and carcinogenesis stimulation [202]. This statin rebound effect can be avoided by, e.g., administering dipyridamole, which inhibits transcription factor SREBP2 [203].
Other enzymes involved in the de novo cholesterol biosynthesis pathway are also promising targets for cancer therapy, such as SQLE. SQLE is influenced by multiple cancer‐promoting pathways, and its upregulation has been associated with various cancer types, including colorectal, breast, ovarian, prostate cancer, hepatocellular carcinoma, squamous lung cancer, and leukemia [28, 204, 205]. Inhibition of SQLE, pharmacologically by inhibitors like terbinafine or by gene silencing, has been shown to reduce cancer cell viability and proliferation. Preclinical studies have demonstrated that terbinafine effectively suppresses tumor growth and decreases cancer cell proliferation in xenograft models. Repurposing SQLE inhibitors, originally developed as antifungal drugs, represents a promising strategy for targeting cholesterol biosynthesis in cancer treatment [206].

LXRs have emerged as promising anticancer targets. The anticancer effects of LXR activation are mediated, as previously discussed, through various pathways besides cholesterol depletion. It has been shown that lowering of cholesterol levels by LXR activation enhances the expression of ABCA1, promoting cellular cholesterol efflux, which exerts antitumor effects in prostate cancer cells [207]. Moreover, in prostate cancer cells, cholesterol depletion via LXR activation disrupts lipid raft composition and interferes with Akt signaling, promoting apoptosis [208]. In glioblastoma cells with mutant activated EGFR, treatment with an LXR agonist induces LDLR degradation and triggers apoptosis [209]. Similarly, in non‐small cell lung carcinoma, where resistance to EGFR tyrosine kinase inhibitors is a challenge, combining these inhibitors with an LXR agonist has demonstrated efficacy in suppressing proliferation and metastasis [210].
However, LXR activation stimulates fatty acid synthesis—a process which potentially promotes cancer. Therefore, a combined therapeutic strategy using LXR agonizts and fatty acid synthase inhibitors has been proposed [43]. LXRs are promising drug targets with the potential to complement statin therapy. The first‐generation LXR agonizts due to pleiotropic actions failed clinical trials. Nonetheless, they highlight the pathway's potential in anticancer strategies. Current efforts focus on overcoming pharmacological limitations, renewing interest in LXRs as therapeutic targets [45, 211].
Targeting cholesterol homeostasis offers a promising avenue for cancer therapy, leveraging both established and novel approaches. Statins, as HMGCR inhibitors, have demonstrated efficacy in reducing tumor growth and metastasis, though challenges such as resistance and off‐target effects remain. Advances in understanding downstream enzymes like SQLE in the cholesterol biosynthesis pathway have revealed additional therapeutic opportunities, with preclinical evidence supporting their potential in reducing cancer cell proliferation. Furthermore, LXRs present a complementary strategy by promoting cholesterol efflux and apoptosis, although their dual effects on fatty acid synthesis need combination therapies for optimal outcomes. Oncologists frequently raise concerns about the clinical significance of statin effects observed in vitro, given the higher concentrations required [212]. This has prompted interest in improving statin efficacy through combination strategies [213, 214], emphasizing the need to explore downstream targets in the mevalonate pathway, particularly enzymes like SQS, which emerge as potential pharmacological targets to decrease cholesterol levels, presenting opportunities for the development of novel non‐statin therapies [18].

2
The Role of SQS in Cancer
SQS is a critical enzyme in the cholesterol biosynthesis pathway [23], localized in the endoplasmatic reticulum [215] and encoded by the FDFT1 gene located in the 8p.22‐23.1 chromosomal region [216]. Its expression is tightly regulated by SREBP transcription factors, which bind to SRE‐like sequences located approximately 150 base pairs above the transcription start site of the enzyme's encoding gene [217]. SQS catalyzes the head‐to‐head condensation of two FPP molecules, producing initially presqualene pyrophosphate, a stable cyclopropylcarbinyl pyrophosphate intermediate, which undergoes heterolytic isomerization and reduction by NADPH, producing eventually squalene (Figure 7). Squalene is the first molecule in the cholesterol biosynthetic pathway that is exclusively intended to produce cholesterol [218] (Figure 1). SQS plays a key role as it directs the FPP metabolite to the sterol branch of the pathway instead of the non‐sterol branch. It is, therefore, a promising target for inhibiting cholesterol biosynthesis, offering advantages over statins by not affecting the production of essential substances produced downstream of HMGR and upstream of SQS, such as dolichols, heme A, ubiquinone, and prenylated proteins [23].
SQS inhibitors are primarily known as potential antihyperlipidemic agents for the control of circulating cholesterol. Recently, they have been applied to investigate their impact on cellular membrane cholesterol content [219]. SQS inhibitors have been shown to regulate various cellular processes linked to neurodegeneration [220, 221, 222], parasite growth [223, 224], and notably, the proliferation and migration of cancer cells [225]. Despite ongoing research on cholesterol's mechanism of action on a cellular level, the evolving evidence positions SQS as a therapeutic target for a variety of diseases [226].
Statins, despite their cost‐effectiveness and their wide use, exhibit limitations, since their inhibition point at HMGCR affects a broad range of molecules [227, 228]. SQS inhibitors, such as zaragozic acid, offer a more specific alternative by targeting cholesterol synthesis downstream of mevalonate formation, preserving isoprenoid production. Moreover, some statin‐induced adverse effects are caused by the downregulation of the non‐sterol branch of the mevalonate pathway, including myotoxicity [229], while coadministration with SQS inhibitors prevents statin‐induced myotoxicity [230].
This targeted inhibition proves advantageous in exploring the role of cholesterol biosynthesis itself and its effect on membrane rafts in cancer cells [231, 232]. Not only does SQS inhibition with zaragozic acid attenuate proliferation and induce apoptosis in prostate cancer cells [231], but it also hinders the growth of RMA lymphoma and Lewis lung carcinoma [233]. These findings underscore SQS as a key player in investigating the intricate connections between cholesterol, membrane rafts, and cancer progression, shedding light on the potential therapeutic approach of SQS inhibition (Figure 8) [226].
2.1
Inhibition of SQS Activity
From a medicinal chemistry perspective, the design and development of small molecules as enzyme inhibitors of SQS has been/still is investigated mainly as an alternative treatment for hyperlipidemia and cardiovascular disease. Main drive, as mentioned above, for this continuing research is the incomplete therapeutic efficacy and/or frequency of side effects exhibited by the mainstream/low‐cost statins.
Inhibitors of SQS (Figure 9, 10, 11, 12, 13) include zaragozic acids (squalestatins), quinuclidine/morpholine derivatives, and 4,1‐benzoxazepine derivatives such as lapaquistat/TAK475. Considering the different modes of action that they exhibit as well as their origin, potent inhibitors of SQS can be categorized as follows [234, 235, 236]:

(1) Structural analogs of FPP substrate: These inhibitors are designed to mimic key components of the FPP substrate. They typically contain isoprene units or other resembling hydrophobic moieties along with a functional group that resembles the diphosphate of FPP [237, 238, 239, 240]. The first such inhibitors consisted of isoprenylphosphinylformates (structure 1, Figure 9) followed by aromatic isosters of the “farnesyl” side chain, such as para‐substituted phenyl analogs 2 and 3 (Figure 9).

(2) Transition‐state analogs: These compounds mimic the transitional states in the interaction between FPP and SQS. By incorporating a basic nitrogen, they mimic the carbocation intermediate, while cyclopropyl amines mimic the cyclopropyl moiety of presqualene [241, 242, 243, 244, 245]. Such inhibitors include N‐(arylalkyl‐1‐)faresylamines (4), phenoxypropylamines (5 and 6), as well as 3’ aromatically substituted quinuclidines like 7‐10 or YM53601 (11) [246] (Figure 9).
In addition, bisphosphonates, aside from being transition‐state analogs, due to their ion‐chelating properties, bind SQS co‐factor Mg2+ [247, 248, 249]. Such compounds with SQS inhibitory activity include 1,1‐biphosphonates 12 and 13, 4‐substituted biphenyl phosphonosulfonates (14), or substituted biaryl ether phosphonosulfonates (15). Other potent inhibitors of this category include YM175 (or else incadronate, 16), bisphosphonate 17, and deshydroxyzoledronate analog 18 [250] (Figure 10).

(3) Natural products: Derived from fungi, zaragozic acids (also known as squalestatins) (structures 19‐21) mimic the binding of presqualene pyrophosphate and inhibit SQS with high potency [251, 252]. Their poor biopharmaceutical properties have led to the development of derivatives [253] such as decarboxy or 4‐deoxy analogs of squalestatins as well as synthetic inhibitors based on their structure, such as A‐87049 (22) [254]. Schizostatin (23) is also fungal‐derived and is supposed to mimic the structure of the two farnesyl chains [255] (Figure 11).

(4) Fusion with antioxidant and/or anti‐inflammatory moieties: By combining SQS inhibitors with antioxidant or anti‐inflammatory components, multiactive molecules were derived using aromatically substituted morpholines (24 and 25), nitric oxide‐releasing moieties (26), benzoxazine/benzothiazine structures (27 and 28), as well as combinations with the phenothiazine moiety (29). Compounds 24, 26, and 29 were shown to reduce atherosclerosis on top of their antihyperlipidemic and other functions [256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267] (Figure 12).

(5) Benzoxazepines: Identified by a wide screening effort, benzoxazepines such as 30 (Figure 13) were revealed as a new class of potent inhibitors that bear the structural moiety of a lactam heterocycle fused with benzene. These structures gave rise through SAR optimization to TAK‐475 (or else lapaquistat, 31), an optimized competitive SQS inhibitor [268, 269]. TAK‐475 underwent clinical trials up to phase III but was discontinued since it appeared to increase hepatic alanine transaminase (ALT) enzymes [270]. New inhibitors in this category, such as DF461 (32) [271] continued to develop, bearing in mind the minimization of adverse effects (Figure 13).

(6) Potent inhibitors were derived structurally based on a 2‐aminobenzhydrol template (33, Figure 13) [272] among which atrop fixed alkoxy‐aminobenzhydrol derivatives (34) were shown to be orally efficacious [272]. More recently, based on SAR studies of the latter alkoxy‐aminobenzhydrol derivatives, 11‐membered templates, such as malonic amide or glycine alkyl type, were developed with potent activity (35 and 36) (Figure 13) [273].

(7) Other different strategies for inhibiting SQS have been investigated, such as:
(a) Developing peptide inhibitors; due to their high specificity, efficacy, low toxicity profile, and well‐tolerability, potential peptide inhibitors were explored via theoretical as well as experimental screening methods. As a result, several ennea‐peptides (e.g CLSPHSMFC), exa‐peptides (e.g., FTACNW), and tetra‐peptides (e.g., SMFC or CKTE) showed promise in effectively inhibiting SQS [274, 275].
(b) Enzyme protein degraders; emerging technologies based on proteolysis‐targeting chimaeras (PROTACs) [276] offer an alternative approach in which, upon drug binding, the target protein is marked for degradation. Although this specific approach has not been so far successful in reducing SQS levels, a small molecule ligand of SQS, KY02111 [277] (37, Figure 13), has been unexpectedly identified to selectively cause SQS to degrade in a proteasome‐dependent manner. As such, KY02111 seems to represent a nonconventional and mechanistically new degrader and the first for SQS.
(c) Exploring potential inhibitors generated from computational studies; with the help of molecular modeling studies, Traditional Chinese Medicine (TCM) and Specs Databases were virtually screened, leading to the identification of Cynarin (38, Figure 13) as a potential SQS inhibitor. Further, novel chemical scaffolds for SQS inhibition were generated via 3D‐QSAR pharmacophore models; virtual screening of chemical databases (Specs and TCM) identified potential candidates from which compounds (such as structure 39, Figure 13) were selected that exhibited potent in vitro activity [278, 279].
The reviews by Kourounakis et al. [261] and Park et al. [235] provide a comprehensive background for structure‐based development of SQS inhibitors.

2.2
The Role of SQS in the Development and Progression of Cancer
SQS is emerging as a promising therapeutic target in cancer due to its elevated expression across numerous cancers, including prostate, breast, ovary, liver, small intestine, bladder, cervix, thyroid, and esophagus, while it seems it can be used as a biomarker in a wide range of cancers. These results are based on the study by Tüzmen et al., where levels of SQS protein and mRNA were evaluated in various types of cancers; in many cases, levels are elevated, with the highest expression observed in liver cancer, where it was fivefold higher than the adjacent normal liver tissue. However, it should be noted that in some other types of cancer, such as stomach, pancreas, uterus, and colorectal cancer, no significant alteration in the expression of SQS was observed, while in several other cancers, as in testicular, kidney, lung, and lymphoma cancers, SQS showed reduced expression. The above phenomenon, in which a cancer‐related gene plays a role that can differ depending on the type of cancer, inhibition ‐ tumor suppressive action ‐ or induction ‐ oncogenic action ‐ is not uncommon [280]. In another study by Hughes et al., analysis of 66 esophageal adenocarcinomas showed consistent SQS amplification and overexpression in 12% of the samples. In addition, a subsequent analysis showed overexpression of SQS mRNA in all amplified esophageal adenocarcinoma cells studied [281]. SQS's notable overexpression in mammospheres and in neuroblastoma sphere‐forming cells (i.e., cancer stem cells with self‐renewal, differentiation, and treatment resistance properties) is associated with advanced cancer stages as well as with reduced relapse‐free survival [282, 283]. Other studies demonstrate that SQS serves as a prognostic marker for colon cancer and promotes cancer cell proliferation [284]. A genomic study further revealed a correlation between high SQS levels in colon cancer and reduced patient survival period [285]. However, another proteogenomic study suggests that overexpression of SQS in colon cancer leads to prolongation of overall survival and progression‐free survival [286]. Tongue squamous cell carcinoma (TSCC), known for its pronounced metastatic nature, is linked to elevated SQS expression that promotes carcinogenesis [287]. In a study by Chattopadhyay and Mallick, the oncogenic roles of SQS were investigated; overexpression of SQS was found to boost TSCC cells' proliferation, migration, and ROS production—the latter being a factor that enhances TSCC cell growth—and a noncoding RNA, called piR‐39980, showed potential inhibitory action to SQS expression [288].
SQS also shows polymorphism, which has been linked to cancer. An association of prostate cancer with the rs2645429 polymorphism of the SQS gene has been observed; this single‐nucleotide polymorphism (C → T replacement) is located six base pairs before the SRE‐1 region, leading to an increase in promoter activity resulting in the transcription induction of the SQS gene. Therefore, patients with this allele have elevated SQS levels and a higher risk of prostate carcinogenesis and metastasis [289]. This polymorphism has also been reported in non‐small cell lung cancer and can be considered a risk factor [290]. Abnormalities in the number of copies of the gene have also been reported in gastric cancer [291], while a mutation in the SQS gene has also been reported in liver metastasis from colorectal cancer [286]. The study by Tüzmen et al. showed an increased expression of specific variants of the SQS gene with alternatively splicing (AS) in cancer tissue samples relative to the corresponding normal tissue adjacent to the tumor, while in a study performed in various cancer cell lines, AS variant 1 (AS‐1) appeared to be predominant. Furthermore, the gene promoter region was studied to find a methylation pattern, but no result was found to justify the variation in SQS expression [280].
The expression of SQS is induced in dividing cells, leading to the conclusion that it significantly contributes to cancer signaling for cell proliferation. SQS is involved via several mechanisms in cancer cell proliferation, one being the increased biosynthesis of cholesterol. SQS seems to control the course of the cell cycle, as inhibition of its expression impairs cell proliferation and maintains cells in the G1 and G2/M phases. However, replenishment with cholesterol revokes the attenuation of the cell cycle, concluding that the action of SQS on the cell cycle is indirect. Thus, inhibition of this enzyme, and consequently, de novo cholesterol synthesis, is an attractive therapeutic strategy for eradicating cancer stem cells as well as inhibiting cancer cell growth [83, 292, 293].
From another point, overexpression of SQS leads to increased squalene levels. Squalene is an antioxidant and emollient for the skin, as it protects cell membranes from lipid peroxidation caused by ROS and is also a major component of skin sebum [294]. Although the role of squalene in cancer has not been thoroughly investigated, it may provide an advantage for cell survival [295, 296]. In Anaplastic Large Cell Lymphoma (ALCL) Anaplastic Lymphoma Kinase‐positive (ALK +) cell lines expression of SQLE is lost, resulting in the accumulation of squalene (normally it is undetectable). In this cancer cell line, squalene alters the lipid profile of the cell and protects it from death by ferroptosis. Squalene, therefore, provides an advantage for growth under oxidative stress. The effect of inhibition of SQS by gene knockdown in mice with ALCL ALK+ was studied. Loss of SQS activity led to a significant reduction in tumor size. Also, SQS gene loss or the use of small SQS inhibitors in cells lacking SQLE led to the sensitization of these cancer cells to ferroptosis (induced by glutathione peroxidase inhibitors). The dependency of cancer cell lines on exogenous intake of a metabolite, in this case cholesterol, since they are unable to synthesize it de novo, is called auxotrophy. Other auxotrophic cancer cells, such as histiocytic lymphoma cancer cell line U‐937 [297] or glioblastoma multiforme cells [298], also show this dependency and are therefore sensitive to decreases in serum cholesterol.
Moreover, targeting SQS and directly inhibiting cholesterol biosynthesis can be used as a therapeutic strategy against chemoresistant acute myeloid leukemia (AML). In AML, exposure to radiotherapy and chemotherapy elevates cholesterol levels, and by blocking cholesterol synthesis through SQS inhibition, the sensitivity of AML cell lines to therapy is heightened [299]. A study conducted by Karakitsou et al. used genome‐scale metabolic models to highlight the potential of SQS inhibition as a promising therapeutic approach for attenuating the growth of chemoresistant AML cells. Upon inhibiting SQS with YM‐53601, the proliferation of chemoresistant AML cell lines, resistant to doxorubicin or cytarabine, was compromised [300].
As previously discussed, tumor‐specific immune responses are influenced by cholesterol and its derivatives, which suppress immune cells within tumors and inhibit their migration to lymphoid organs [100]. SQS inhibition by zaragosic acids does not affect the formation of isoprenoids crucial for the maturation of proteins involved in immune cell function. Many immune cells rely on protein prenylation for essential processes, including motility, activation, proliferation, and antigen uptake [301]. Zaragozic acid significantly suppresses in vivo tumor growth of RMA lymphoma and Lewis lung carcinoma (LLC) and depends on a functional immune system. In contrast, zoledronic acid, which inhibits FPP synthesis and consequently isoprenoid formation, fails to control LLC tumor growth. Toxicity evaluation of zaragozic acid indicates no hepatotoxicity even in high antitumor doses; however, comprehensive information on bioavailability and pharmacological features is needed before clinical translation of these compounds. Furthermore, the combination of zaragozic acid with immunotherapy effectively inhibits tumor growth, and this synergistic action significantly prolongs the overall survival of mice with LLC or RMA lymphoma. These findings underscore the potential of zaragozic acids and SQS inhibition as a promising therapeutic approach in antitumor immune responses and need further exploration in combination with immunotherapy for cancer treatment [233].
2.2.1
The Role of SQS in Prostate Cancer
The study of the relationship between SQS and prostate cancer is quite interesting. By analyzing the genomes of prostate cancer patients, a correlation with a peak marker (D8S550) was discovered in a chromosomal region (8p23) where the SQS gene is located. A specific genotype (GA + AA genotype of the rs2645429 polymorphism, GG: wild type, GA: heterozygous, AA: mutant) was found to increase the risk of prostate cancer. This AA genotype of the rs2645429 mutation causes increased activation of the SQS gene promoter. Furthermore, the role of SQS in the proliferation of prostate cancer cells was investigated; initially, the SQS gene was silenced by siRNA, resulting in inhibition of proliferation in both LNCaP and PC3 prostate cancer cell lines. Subsequently, inhibition of SQS by TAK‐475 caused a significant dose‐dependent inhibition of LNCaP and PC3 cell proliferation. Lastly, prostate biopsy showed that the expression of the SQS gene was high in prostate cancer tissues and was even higher in aggressive types of cancers [289].
In the research by Brusselmans et al., androgens have been shown to induce SREBP activation in androgen‐dependent prostate cancer cells. Despite adequate cholesterol levels, they lead to increased expression of mevalonate pathway enzymes, including SQS. As a result, androgens led to increased levels of de novo synthesized cholesterol in lipid rafts (5‐fold increase), whereas cholesterol levels outside of lipid rafts were less affected (2‐fold increase). Inhibition of SQS by RNAi blocked cholesterol biosynthesis in LNCaP cells and caused a 50% reduction in cholesterol content of lipid rafts, without any effect on the non‐sterol branch of the mevalonate pathway. This led to cytotoxicity and to significant inhibition of LNCaP cell proliferation—effects that were reversible upon supplying exogenous cholesterol. Furthermore, administration of the SQS inhibitor zaragosic acid led to a 60% reduction in cholesterol content of lipid rafts, inducing cytotoxicity and growth inhibition of LNCaP cells. In conclusion, androgens play a regulatory role in SQS expression within prostate cancer cell lines [231] and in support of this, a study conducted by Fukuma et al. demonstrated that after a 6‐month period of androgen deprivation therapy, SQS levels were significantly decreased [289].
Taken together, interference in the sterol branch of the mevalonate pathway by SQS inhibitors is a potentially new approach for treating and/or preventing prostate cancer. Finally, it is worth mentioning that because solid tumors have a limited nutrient supply, they are more dependent on de novo cholesterol synthesis, making SQS in this case a very interesting antineoplastic target [231, 289].

2.3
The Role of SQS in Metastasis
In a study conducted by Yang et al. [30] on lung cancer patients, 27 out of the 30 patients showed significantly increased SQS expression in cancerous tissue compared to neighboring healthy tissue. The results showed that SQS was associated with advanced stages of lung cancer, increased tumor size, metastasis in regional lymph nodes, distant metastasis, and relapse. Upregulation of SQS in patients with lung cancer is remarkably associated with shorter disease‐free as well as overall survival and with worse prognosis, compared to patients with low levels of SQS. Additionally, this study revealed that in lung cancer cells with a high invading tendency, eight genes involved in cholesterol biosynthesis are upregulated, including HMG‐CoA synthetase 1, SQS, and SQLE. Inhibition of SQS in metastatic lung cancer cells, either with zaragozic acid or by gene knockdown, causes a significant reduction of mobility and invasion ability in vitro, as well as decreased tumor burden and metastasis formation in vivo. Notably, inhibition of HMGCR and of HMG‐CoA synthetase 1 had a minor effect on lung cancer cell invasiveness in vitro in comparison with SQS inhibition. To confirm the role of cholesterol in the above findings, the effect of mevalonate pathway inhibition downstream of SQS was studied; indeed, it was confirmed that cholesterol itself is associated with the progression of lung cancer. Moreover, forced overexpression of SQS in cells with low invasion tendency increases their migration and invasion abilities, resulting in an increased number of detectable lung metastases. But what further mechanisms may be involved? SQS upregulation causes increased cholesterol levels, which enrich lipid rafts with tumor necrosis factor receptor 1 (TNFR1), leading to NF‐κB activation [302, 303]. This activation results in increased expression of MMP proteinases, which are in turn associated with metastatic processes but also with growth and progression of lung cancer [30, 304]. These findings support the potential of targeting SQS as a promising therapeutic strategy for combating malignant lung cancer.
Besides TNFR1, SQS is involved in metastasis through osteopontin, the expression of which is induced by elevated cholesterol biosynthesis. SQS overexpression causes an increase in osteopontin expression, which then induces phosphorylation of Src, ERK, and Akt kinases, thereby increasing MMP‐1 expression and thus inducing metastasis of lung cancer cells. High osteopontin expression is associated with smoking, lymph node metastasis, advanced stages of cancer, poor prognosis, and reduced overall survival in patients with lung cancer. Osteopontin also induces metastasis in various types of cancers as it participates in processes such as cell adhesion, chemotaxis, invasion, migration, and anchorage‐independent growth of tumor cells. Osteopontin is upregulated in lung antigen‐presenting cells and is essential for IL‐17A production, which in turn regulates lung cancer growth. Finally, osteopontin interacts directly with membrane receptors such as EGFR, VEGF, CD44, and integrins, thus promoting metastasis also via this mode of action. Knockdown of SQS leads to disruption of lipid rafts, resulting in shedding of the overexpressed CD44 receptor in lung cancer cells and in inhibition of tumor cell migration [305].
These findings suggest that SQS drives lung cancer metastasis through increasing the levels of cholesterol, which exclusively intermediates the above molecular mechanisms.

3
Conclusions
Cholesterol plays a complex role in carcinogenesis, tumor growth, and metastasis through various mechanisms. On one hand, oncogenic pathways increase intracellular cholesterol levels, and on the other hand, cholesterol and its derivatives further support cancer cell proliferation, migration, and metastasis. A key mechanism by which cholesterol supports cancer is through lipid raft formation, which serves as a signaling platform for cancer‐related receptors and pathways, along with contributing to cancer cell mobility and invasion.
Several studies indicate that inhibition of cholesterol biosynthesis suppresses various cancer actions. Cell cycle is disrupted, steroidogenesis is impeded, immune response against cancer cells can be induced, and resistance to chemotherapy can be reversed. Moreover, cholesterol depletion from lipid rafts inhibits oncogenic signaling. Cholesterol biosynthesis inhibition becomes, thus, an attractive target for anticancer therapy. Indeed, statins, the most widely used inhibitors of cholesterol biosynthesis, have been extensively studied for their anticancer effect. However, their action on HMGCR is early in the mevalonate pathway, affecting multiple molecules. Therefore, their action cannot be attributed solely to the lowering of cholesterol levels, and as a matter of fact, inhibition of the non‐sterol pathway, instead of cholesterol biosynthesis, has been mainly associated with their anticancer effects. To clearly understand the impact of lowering intracellular cholesterol levels and their derivatives in cancer, targeting SQS appears more promising. SQS directs the mevalonate pathway towards cholesterol biosynthesis instead of the non‐sterol branch, and its dysregulation is linked to various cancers. Through SQS inhibition, cancer growth is hindered, and cancer cells are sensitized to ferroptosis, while inhibition of SQS also results in a decrease in the migration and metastatic tendency. Notably, SQS inhibition lowers cholesterol without affecting protein prenylation, which is essential for immune cells' function against cancer cells.
Research suggests that SQS inhibition, especially in combination with immunotherapy or chemotherapy, could be a valuable strategy to combat cancer and reduce treatment resistance. Further research into SQS inhibition across various cancer types holds potential for developing novel therapeutic approaches in cancer therapy.

Supporting information
Supplementary Information – Not for Review.