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Cholesterol is essential for cells to grow and proliferate. Normal mammalian cells meet their need for cholesterol through its uptake or de novo synthesis 1 , but the extent to which cancer cells rely on each of these pathways remains poorly understood. Here, using a competitive proliferation assay on a pooled collection of DNA-barcoded cell lines, we identify a subset of cancer cells that is auxotrophic for cholesterol and thus highly dependent on its uptake. Through metabolic gene expression analysis, we pinpoint the loss of squalene monooxygenase expression as a cause of cholesterol auxotrophy, particularly in ALK + anaplastic large cell lymphoma (ALCL) cell lines and primary tumours. Squalene monooxygenase catalyses the oxidation of squalene to 2,3-oxidosqualene in the cholesterol synthesis pathway and its loss results in accumulation of the upstream metabolite squalene, which is normally undetectable. In ALK + ALCLs, squalene alters the cellular lipid profile and protects cancer cells from ferroptotic cell death, providing a growth advantage under conditions of oxidative stress and in tumour xenografts. Finally, a CRISPR-based genetic screen identified cholesterol uptake by the low-density lipoprotein receptor as essential for the growth of ALCL cells in culture and as patient-derived xenografts. This work reveals that the cholesterol auxotrophy of ALCLs is a targetable liability and, more broadly, that systematic approaches can be used to identify nutrient dependencies unique to individual cancer types. The authors find that loss of squalene monooxygenase expression alters the lipid metabolism of cancer cells, which confers protection from ferroptotic cell death and thus promotes tumour growth.

Squalene epoxidase (SQLE), also known as squalene monooxygenase, catalyzes the stereospecific conversion of squalene to 2,3( S )-oxidosqualene, a key step in cholesterol biosynthesis. SQLE inhibition is targeted for the treatment of hypercholesteremia, cancer, and fungal infections. However, lack of structure-function understanding has hindered further progression of its inhibitors. We have determined the first three-dimensional high-resolution crystal structures of human SQLE catalytic domain with small molecule inhibitors (2.3 Å and 2.5 Å). Comparison with its unliganded state (3.0 Å) reveals conformational rearrangements upon inhibitor binding, thus allowing deeper interpretation of known structure-activity relationships. We use the human SQLE structure to further understand the specificity of terbinafine, an approved agent targeting fungal SQLE, and to provide the structural insights into terbinafine-resistant mutants encountered in the clinic. Collectively, these findings elucidate the structural basis for the specificity of the epoxidation reaction catalyzed by SQLE and enable further rational development of next-generation inhibitors. Squalene epoxidase (SQLE) is a key enzyme in cholesterol biosynthesis and is a target for hypercholesteremia and cancer drug development. Here the authors present the crystal structures of the human SQLE catalytic domain alone and bound with small molecule inhibitors, which will facilitate the development of next-generation SQLE inhibitors.

The consumption of sweeteners, natural as well as synthetic sugars, is implicated in an array of modern-day health problems. Therefore, natural nonsugar sweeteners are of increasing interest. We identify here the biosynthetic pathway of the sweet triterpenoid glycoside mogroside V, which has a sweetening strength of 250 times that of sucrose and is derived from mature fruit of luohan-guo (Siraitia grosvenorii, monk fruit). A whole-genome sequencing of Siraitia, leading to a preliminary draft of the genome, was combined with an extensive transcriptomic analysis of developing fruit. A functional expression survey of nearly 200 candidate genes identified the members of the five enzyme families responsible for the synthesis of mogroside V: squalene epoxidases, triterpenoid synthases, epoxide hydrolases, cytochrome P450s, and UDP-glucosyltransferases. Protein modeling and docking studies corroborated the experimentally proven functional enzyme activities and indicated the order of the metabolic steps in the pathway. A comparison of the genomic organization and expression patterns of these Siraitia genes with the orthologs of other Cucurbitaceae implicates a strikingly coordinated expression of the pathway in the evolution of this species-specific and valuable metabolic pathway. The genomic organization of the pathway genes, syntenously preserved among the Cucurbitaceae, indicates, on the other hand, that gene clustering cannot account for this novel secondary metabolic pathway.

Prostate cancer (PCa) shows strong dependence on the androgen receptor (AR) pathway. Here, we show that squalene epoxidase (SQLE), an enzyme of the cholesterol biosynthesis pathway, is overexpressed in advanced PCa and its expression correlates with poor survival. SQLE expression is controlled by micro-RNA 205 (miR-205), which is significantly downregulated in advanced PCa. Restoration of miR-205 expression or competitive inhibition of SQLE led to inhibition of de novo cholesterol biosynthesis. Furthermore, SQLE was essential for proliferation of AR-positive PCa cell lines, including abiraterone or enzalutamide resistant derivatives, and blocked transactivation of the AR pathway. Inhibition of SQLE with the FDA approved antifungal drug terbinafine also efficiently blocked orthotopic tumour growth in mice. Finally, terbinafine reduced levels of prostate specific antigen (PSA) in three out of four late-stage PCa patients. These results highlight SQLE as a therapeutic target for the treatment of advanced PCa. Cholesterol metabolism is involved in the progression of aggressive prostate cancer (PCa). Here the authors show that miR-205 downregulation promotes cholesterol synthesis and androgen receptor signalling in PCa through enhancing the expression of the rate-limiting enzyme of cholesterol synthesis, squalene epoxidase.

Altered cholesterol metabolism has been linked to a poor prognosis in various types of cancer. Cholesterol oxidation can lead to lipid peroxidation, membrane damage, and cell death. Ferroptosis is a regulated form of cell death characterized by the accumulation of lipid peroxides, which significantly inhibits the growth of ovarian cancer cells. SQLE is the primary enzyme responsible for catalyzing cholesterol lipid synthesis and is notably expressed in ovarian cancer tissues and cells. This study aims to investigate the role of squalene monooxygenase (SQLE) in ferroptosis in ovarian cancer. The protein and mRNA expression of SQLE was assessed using qRT-PCR, Western Blot, and immunohistochemistry. The association between SQLE and ferroptosis was demonstrated through analysis of TCGA and GTEx databases, TMT protein sequencing, as well as validation by qRT-PCR, Western Blot, immunofluorescence, ROS detection, and lipid peroxide detection. Animal experiments further confirmed the relationship between SQLE and ferroptosis in ovarian cancer. The protein and mRNA expression of SQLE was found to be upregulated in both ovarian cancer tissues and cell lines. Decreased SQLE expression led to ferroptosis in ovarian cancer cells, thereby increasing their sensitivity to ferroptosis inducers. Our research demonstrates that SQLE is significantly upregulated in both ovarian cancer tissues and cells. The overexpression of SQLE in ovarian cancer may facilitate tumorigenesis by conferring resistance to ferroptosis, thus shedding light on potential novel therapeutic strategies for ovarian cancer.

ObjectiveSqualene epoxidase (SQLE) promotes metabolic dysfunction-associated steatohepatitis-associated hepatocellular carcinoma (MASH-HCC), but its role in modulating the tumour immune microenvironment in MASH-HCC remains unclear.DesignWe established hepatocyte-specific Sqle transgenic (tg) and knockout mice, which were subjected to a choline-deficient high-fat diet plus diethylnitrosamine to induce MASH-HCC. SQLE function was also determined in orthotopic and humanised mice. Immune landscape alterations of MASH-HCC mediated by SQLE were profiled by single-cell RNA sequencing and flow cytometry.ResultsHepatocyte-specific Sqle tg mice exhibited a marked increase in MASH-HCC burden compared with wild-type littermates, together with decreased tumour-infiltrating functional IFN-γ+ and Granzyme B+ CD8+ T cells while enriching Arg-1+ myeloid-derived suppressor cells (MDSCs). Conversely, hepatocyte-specific Sqle knockout suppressed tumour growth with increased cytotoxic CD8+ T cells and reduced Arg-1+ MDSCs, inferring that SQLE promotes immunosuppression in MASH-HCC. Mechanistically, SQLE-driven cholesterol accumulation in tumour microenvironment underlies its effect on CD8+ T cells and MDSCs. SQLE and its metabolite, cholesterol, impaired CD8+ T cell activity by inducing mitochondrial dysfunction. Cholesterol depletion in vitro abolished the effect of SQLE-overexpressing MASH-HCC cell supernatant on CD8+ T cell suppression and MDSC activation, whereas cholesterol supplementation had contrasting functions on CD8+ T cells and MDSCs treated with SQLE-knockout supernatant. Targeting SQLE with genetic ablation or pharmacological inhibitor, terbinafine, rescued the efficacy of anti-PD-1 treatment in MASH-HCC models.ConclusionSQLE induces an impaired antitumour response in MASH-HCC via attenuating CD8+ T cell function and augmenting immunosuppressive MDSCs. SQLE is a promising target in boosting anti-PD-1 immunotherapy for MASH-HCC.

Cholesterol synthesis is both energy- and oxygen-intensive, yet relatively little is known of the regulatory effects of hypoxia on pathway enzymes. We previously showed that the rate-limiting and first oxygen-dependent enzyme of the committed cholesterol synthesis pathway, squalene monooxygenase (SM), can undergo partial proteasomal degradation that renders it constitutively active. Here, we show hypoxia is a physiological trigger for this truncation, which occurs through a two-part mechanism: (1) increased targeting of SM to the proteasome via stabilization of the E3 ubiquitin ligase MARCHF6 and (2) accumulation of the SM substrate, squalene, which impedes the complete degradation of SM and liberates its truncated form. This preserves SM activity and downstream pathway flux during hypoxia. These results uncover a feedforward mechanism that allows SM to accommodate fluctuating substrate levels and may contribute to its widely reported oncogenic properties. Cells need cholesterol to work properly but too much cholesterol is harmful and can contribute to atherosclerosis (narrowing of blood vessels), cancer and other diseases. Cells therefore carefully control the activity of the enzymes that are involved in making cholesterol, including an enzyme known as squalene monooxygenase. When the level of cholesterol in a cell rises, a protein called MARCHF6 adds molecules of ubiquitin to squalene monooxygenase. These molecules act as tags that direct the enzyme to be destroyed by a machine inside cells, known as the proteasome, thereby preventing further (unnecessary) production of cholesterol. Previous studies found that squalene monooxygenase is sometimes only partially broken down to make a shorter (truncated) form of the enzyme that is permanently active, even when the level of cholesterol in the cell is high. However, it was unclear what triggers this partial breakdown. The process of making cholesterol uses a lot of oxygen, yet many cancer cells thrive in tumours with low levels of oxygen. Here, Coates et al. used biochemical and cell biology approaches to study the effect of low oxygen levels on the activity of squalene monooxygenase in human cells. The experiments revealed that low oxygen levels trigger squalene monooxygenase to be partially degraded to make the truncated form of the enzyme. Firstly, MARCHF6 accumulates and adds ubiquitin to the enzyme to accelerate its delivery to the proteasome. Secondly, as the proteasome starts to degrade the enzyme, a build-up of squalene molecules impedes further breakdown of the enzyme. This mechanism preserves squalene monooxygenase activity when oxygen levels drop in cells, which may compensate for temporary oxygen shortfalls and allow cells to continue to make cholesterol. Squalene monooxygenase is overactive in individuals with a wide variety of diseases including fatty liver and prostate cancer. Drugs that block squalene monooxygenase activity have been shown to stop cancer cells from growing, but unfortunately these drugs are also toxic to mammals. These findings suggest that reducing the activity of squalene monooxygenase in more subtle ways, such as stopping it from being partially degraded, may be a more viable treatment strategy for cancer and other diseases associated with high levels of cholesterol.

Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers. Cancer cells are metabolically adaptable and the identification of specific vulnerabilities is challenging. Here the authors identify a subset of neuroendocrine cell lines exquisitely sensitive to inhibition of SQLE, an enzyme in the cholesterol biosynthetic pathway, due to the toxic accumulation of pathway intermediate squalene.

Colon adenocarcinoma (COAD) is one of the most prevalent malignancies, with poor prognosis and lack of effective treatment targets. Squalene synthase (FDFT1) is an upstream enzyme of squalene epoxidase (SQLE) in cholesterol biosynthesis. In a previous study, we revealed that SQLE promotes colon cancer cell proliferation in vitro and in vivo. Here, we investigate the prognostic value of FDFT1 in stage I‐III COAD and explore the potential underlying mechanisms. Squalene synthase was significantly upregulated in stage I‐III COAD and positively correlated with poor differentiation and advanced tumor stage. High expression of FDFT1 was an independent predictor of overall and relapse‐free survival, and the nomograms based on FDFT1 could effectively identify patients at high risk of poor outcome. Squalene synthase accelerated colon cancer cell proliferation and promoted tumor growth. Lack of FDFT1 resulted in accumulating NAT8 and D‐pantethine to lower reactive oxygen species levels and inhibit colon cancer cell proliferation. Moreover, the combined inhibition of FDFT1 and SQLE induced a greater suppressive effect on cell proliferation and tumor growth than single inhibition. Taken together, these results indicate that FDFT1 predicts poor prognosis in stage I‐III COAD and has the tumor‐promoting effect on COAD through regulating NAT8 and D‐pantethine. Targeting both FDFT1 and SQLE is a more promising therapy than their single inhibition for stage I‐III COAD. Squalene synthase (FDFT1) induces accumulation of reactive oxygen species and promotes colon cancer cell proliferation through upregulation of D‐pantethine and NAT8. In addition, FDFT1 synergizes squalene epoxidase to facilitate colon adenocarcinoma progression.

Squalene epoxidase (SQLE) is a crucial enzyme in the cholesterol-biosynthesis pathway and a promising target for treating cholesterol-related disorders. This study aimed to repurpose eighteen clinically approved phenothiazine antipsychotics as competitive SQLE inhibitors by integrating structure-based virtual screening, 200 ns molecular-dynamics simulations, MM/PBSA binding-energy calculations and in vitro enzyme assays. We first screened the 18 derivatives using molecular docking, structural/pharmacological diversity and ADMET analysis. Six compounds—ethopropazine, periciazine, piperacetazine, dixyrazine, fluphenazine and trifluoperazine—were prioritized for detailed MD simulations and MM/PBSA evaluation. Potential-energy-landscape analysis revealed that ethopropazine, periciazine and piperacetazine formed the most stable enzyme–ligand complexes, each occupying well-defined energy wells. Corresponding ΔG total values of − 27.05 ± 2.10 kcal mol⁻¹, − 27.84 ± 1.67 kcal mol⁻¹ and − 26.94 ± 1.82 kcal mol⁻¹ indicated high binding affinities. In   vitro assays confirmed potent SQLE inhibition, with IC₅₀ values of 1.69 ± 0.06 µM, 1.55 ± 0.13 µM and 1.44 ± 0.04 µM, respectively; kinetic studies established competitive inhibition with K i values of 0.65–0.69 µM. The strong correlation between computational predictions and experimental data underscores the effectiveness of our integrated approach and identifies ethopropazine, periciazine and piperacetazine as promising lead compounds for further optimization and pre-clinical development as SQLE inhibitors.