Cholesterol Biosynthetic Pathway Research Articles

Lipid metabolism: a central modulator of RORt-mediated Th17 cell differentiation.

Among the T helper cell subsets, Th17 cells contribute to the development of various inflammatory and autoimmune diseases, including psoriasis, rheumatoid arthritis, inflammatory bowel disease, steroid-resistant asthma, and multiple sclerosis. Retinoid-related orphan receptor gamma t (RORγt), a nuclear hormone receptor, serves as a master transcription factor for Th17 cell differentiation. Recent findings have shown that modulating the metabolic pathway is critical for Th17 cell differentiation, particularly through the engagement of de novo lipid biosynthesis. Suppression of lipid biosynthesis, either through the pharmacological inhibition or gene deletion of related enzymes in CD4+ T cells, results in significant impairment of Th17 cell differentiation. Mechanistic studies indicate that metabolic fluxes through both the fatty acid and cholesterol biosynthetic pathways have a pivotal role in the regulation of RORγt activity through the generation of endogenous RORγt lipid ligands. This review discusses recent discoveries highlighting the importance of lipid metabolism in Th17 cell differentiation and function, as well as exploring specific molecular pathways involved in RORγt activation through cellular lipid metabolism. We further elaborate on a pioneering therapeutic approach to improve inflammatory and autoimmune disorders via the inhibition of RORγt.

Identification of Anticancer Enzymes and Biomarkers for Hepatocellular Carcinoma through Constraint-Based Modeling.

Hepatocellular carcinoma (HCC) results in the abnormal regulation of cellular metabolic pathways. Constraint-based modeling approaches can be utilized to dissect metabolic reprogramming, enabling the identification of biomarkers and anticancer targets for diagnosis and treatment. In this study, two genome-scale metabolic models (GSMMs) were reconstructed by employing RNA sequencing expression patterns of hepatocellular carcinoma (HCC) and their healthy counterparts. An anticancer target discovery (ACTD) framework was integrated with the two models to identify HCC targets for anticancer treatment. The ACTD framework encompassed four fuzzy objectives to assess both the suppression of cancer cell growth and the minimization of side effects during treatment. The composition of a nutrient may significantly affect target identification. Within the ACTD framework, ten distinct nutrient media were utilized to assess nutrient uptake for identifying potential anticancer enzymes. The findings revealed the successful identification of target enzymes within the cholesterol biosynthetic pathway using a cholesterol-free cell culture medium. Conversely, target enzymes in the cholesterol biosynthetic pathway were not identified when the nutrient uptake included a cholesterol component. Moreover, the enzymes PGS1 and CRL1 were detected in all ten nutrient media. Additionally, the ACTD framework comprises dual-group representations of target combinations, pairing a single-target enzyme with an additional nutrient uptake reaction. Additionally, the enzymes PGS1 and CRL1 were identified across the ten-nutrient media. Furthermore, the ACTD framework encompasses two-group representations of target combinations involving the pairing of a single-target enzyme with an additional nutrient uptake reaction. Computational analysis unveiled that cell viability for all dual-target combinations exceeded that of their respective single-target enzymes. Consequently, integrating a target enzyme while adjusting an additional exchange reaction could efficiently mitigate cell proliferation rates and ATP production in the treated cancer cells. Nevertheless, most dual-target combinations led to lower side effects in contrast to their single-target counterparts. Additionally, differential expression of metabolites between cancer cells and their healthy counterparts were assessed via parsimonious flux variability analysis employing the GSMMs to pinpoint potential biomarkers. The variabilities of the fluxes and metabolite flow rates in cancer and healthy cells were classified into seven categories. Accordingly, two secretions and thirteen uptakes (including eight essential amino acids and two conditionally essential amino acids) were identified as potential biomarkers. The findings of this study indicated that cancer cells exhibit a higher uptake of amino acids compared with their healthy counterparts.

Lipid metabolism is dysregulated in endocrine glands upon autoimmune demyelination

Disturbance in neuroendocrine signaling has been consistently documented in multiple sclerosis (MS), a chronic autoimmune disorder of the central nervous system (CNS) representing the main cause of non-traumatic brain injury among young adults. In fact, MS patients display altered hormonal levels and psychiatric symptoms along with the pathologic hallmarks of the disease, which include demyelination, neuroinflammation and axonal injury. In addition, we have recently shown that extensive transcriptional changes take place in the hypothalamus of mice upon the MS model experimental autoimmune encephalomyelitis (EAE). We also detected structural and functional aberrancies in endocrine glands of EAE animals. Specifically, we described the hyperplasia of adrenal glands and the atrophy of ovaries at disease peak. To further expand the characterization of these phenotypes, here we profiled the transcriptomes of both glands by means of RNA-seq technology. Notably, we identified fatty acid and cholesterol biosynthetic pathways as the most dysregulated molecular processes in adrenals and ovaries, respectively. Furthermore, we demonstrated that key genes encoding neuropeptides and hormone receptors undergo distinct expression dynamics in the hypothalamus along disease progression. Altogether, our results corroborate the dysfunction of the neuroendocrine system as a major pathological event of autoimmune demyelination and highlight the crosstalk between the CNS and the periphery in mediating such disease phenotypes.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Cholesterol biosynthetic pathway induces cellular senescence through ERRα

Cellular senescence is a cell program induced by various stresses that leads to a stable proliferation arrest and to a senescence-associated secretory phenotype. Accumulation of senescent cells during age-related diseases participates in these pathologies and regulates healthy lifespan. Recent evidences point out a global dysregulated intracellular metabolism associated to senescence phenotype. Nonetheless, the functional contribution of metabolic homeostasis in regulating senescence is barely understood. In this work, we describe how the mevalonate pathway, an anabolic pathway leading to the endogenous biosynthesis of poly-isoprenoids, such as cholesterol, acts as a positive regulator of cellular senescence in normal human cells. Mechanistically, this mevalonate pathway-induced senescence is partly mediated by the downstream cholesterol biosynthetic pathway. This pathway promotes the transcriptional activity of ERRα that could lead to dysfunctional mitochondria, ROS production, DNA damage and a p53-dependent senescence. Supporting the relevance of these observations, increase of senescence in liver due to a high-fat diet regimen is abrogated in ERRα knockout mouse. Overall, this work unravels the role of cholesterol biosynthesis or level in the induction of an ERRα-dependent mitochondrial program leading to cellular senescence and related pathological alterations.

Effects of Long-Term High-Ergosterol Intake on the Cholesterol and Vitamin D Biosynthetic Pathways of Rats Fed a High-Fat and High-Sucrose Diet.

Dyslipidemia is a lifestyle-related (physical inactivity or obesity) disease; therefore, dietary foods that can easily be consumed in daily life is important to prevent dyslipidemia. Ergosterol, a precursor of vitamin D2, is a fungal sterol present in the membranes of edible mushrooms and other fungi. Ergosterol is converted to brassicasterol by 7-dehydrocholesterol reductase (DHCR7), a cholesterol biosynthesis enzyme that converts 7-dehydrocholesterol (a precursor of vitamin D3) into cholesterol. Previously, we reported that ergosterol increases 7-dehydrocholesterol, decreases cholesterol levels by competitive effect of DHCR7, and reduces DHCR7 mRNA and protein levels in human HepG2 hepatoma cells. Here, we investigated the effects of long-term high ergosterol intake on the cholesterol, vitamin D2, and D3 biosynthetic pathways of rats fed a high-fat and high-sucrose (HFHS) diet using GC-MS and LC with tandem mass spectrometry. In HFHS rats, oral ergosterol administration for 14 weeks significantly decreased plasma low-density lipoprotein cholesterol, total bile acid, and cholesterol precursor (squalene and desmosterol) levels and increased 7-dehydrocholesterol levels compared to HFHS rats without ergosterol. Ergosterol, brassicasterol, and vitamin D2 were detected, cholesterol levels were slightly decreased, and levels of vitamin D3 and its metabolites were slightly increased in rats fed HFHS with ergosterol. These results showed that ergosterol increased vitamin D2 levels, inhibited the cholesterol biosynthetic pathway, and possibly promoted vitamin D3 biosynthesis in vivo. Therefore, daily ergosterol intake may aid in the prevention of dyslipidemia.

CDK12/13: A Promising New Therapeutic Target in ALL

Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy in children and young adults. Upfront resistance to glucocorticoids used as first-line treatment in most regimens confers a poor prognosis for patients with ALL. Of patients who relapse, only half achieve long-term survival. Here we demonstrate that inhibition of cyclin dependant kinases 12 and 13 (CDK12 & CDK13), which are modulators of RNA polymerase II, has a robust synergistic anti-leukemia effect with dexamethasone, in both B-ALL and T-ALL cell lines as well as primary B-ALL patient-derived bone marrow samples. To understand the mechanism underlying the steroid resistance in ALL, we first reanalyzed shRNA screen performed by Poulard et al. This revealed that CDK13 silencing sensitized leukemic cells to dexamethasone. CDK12 is located on chromosome 17q12, containing oncogenic features and genetic alterations in various tumors. CDK12/13 inhibitor exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft mouse model. It was shown that the inhibition resensitizes HER2+ breast cancers to HER2-tyrosine kinase inhibitors via suppressing PI3K/AKT in patient-derived organoids in vitro and cell-derived xenograft or patient-derived xenograft in vivo. Forgoing research demonstrates that CDK12/13 inhibition silences transcription of DNA damage response (DDR) genes. In our study, however, we haven't observed a rise in γH2AX, a marker of DNA damage, after CDK12/13 inhibition in ALL cell lines. What we did note was a marked enhancement expression of BIM and SGK1, steroid responsive genes and pro-apoptotic factors, after CDK12/13 inhibition (Figure A). ALL cell lines exhibited high susceptibility to CDK12/13 inhibition with two chemically divergent inhibitors, SR-4835 and THZ-531. We also showcased their synergistic interactions with dexamethasone using flow cytometry and synergyfinder ZIP score analysis (Figure B). Moreover, in the GSEA comparison of ALL cell lines with and without synergy between CDK12/13 inhibitors and dexamethasone, our data underscored significant enrichment in cholesterol biosynthesis-related genesets in cell lines manifesting this synergistic effect. On the other hand, we observed resistance to SR-4835 in cell lines with high enrichment scores in NOTCH, a signaling pathway implicated in about 80% of relapsed T cell ALL/LBL cases. Additionally, new data on mice xenotransplanted with B-ALL CNS-derived patient cells, link upregulated cholesterol biosynthetic pathways to nervous system involvement in childhood ALL. Upon administering CDK12/13 inhibitors, we observed a cell cycle phase shift resulting in G1 arrest and increased apoptosis, whereas human peripheral blood mononuclear cells (PBMCs) from healthy individuals remained unaffected by this treatment. As we continue to decode the pathways through which CDK12/13 inhibitors render ALL cells more susceptible to dexamethasone, our next step includes silencing CDK12/13 using short hairpin RNA models in cell lines and consequently their RNA sequencing. This approach may mitigate the influence of secondary pathways, paving the way for enhanced treatment strategies for relapsed ALL by unmasking the underlying molecular events.

Toxicological Mechanisms and Potencies of Organophosphate Esters in KGN Human Ovarian Granulosa Cells as Revealed by High-throughput Transcriptomics.

Despite the growing number of studies reporting potential risks associated with exposure to organophosphate esters (OPEs), their molecular mechanisms of action remain poorly defined. We used the high-throughput TempO-Seq™ platform to investigate the effects of frequently detected OPEs on the expression of ∼3000 environmentally responsive genes in KGN human ovarian granulosa cells. Cells were exposed for 48 h to one of five OPEs (0.1 to 50 μM): tris(methylphenyl) phosphate (TMPP), isopropylated triphenyl phosphate (IPPP), tert-butylphenyl diphenyl phosphate (BPDP), triphenyl phosphate (TPHP), or tris(2-butoxyethyl) phosphate (TBOEP). The sequencing data indicate that four OPEs induced transcriptional changes, whereas TBOEP had no effect within the concentration range tested. Multiple pathway databases were used to predict alterations in biological processes based on differentially expressed genes. At lower concentrations, inhibition of the cholesterol biosynthetic pathway was the predominant effect of OPEs; this was likely a consequence of intracellular cholesterol accumulation. At higher concentrations, BPDP and TPHP had distinct effects, primarily affecting pathways involved in cell cycle progression and other stress responses. Benchmark concentration (BMC) modelling revealed that BPDP had the lowest transcriptomic point of departure. However, in vitro to in vivo extrapolation modeling indicated that TMPP was bioactive at lower concentrations than the other OPEs. We conclude that these new approach methodologies provide information on the mechanism(s) underlying the effects of data-poor compounds and assist in the derivation of protective points of departure for use in chemical read-across and decision-making.

THU542 Inhibiting Oxidosqualene Cyclase Suppresses Cancer Stem-like Cells And Growth Of Epithelial Ovarian Cancer Cells In Vitro And In Vivo

Abstract Disclosure: Y. Liang: None. K.P. Nephew: None. S.M. Hyder: None. Epithelial ovarian cancer (EOC) is an aggressive and deadly disease and despite concerted efforts to develop new strategies for preventing and treating the disease, almost 25,000 new cases are reported each year in the United States. Unfortunately, patients with ovarian cancer have poor prognosis due to the emergence of drug resistant cells and subsequent metastasis and have the highest mortality rate of all known gynecological malignancies. Standard treatments include systemic high-dose toxic chemotherapeutic drugs, but drug-resistance almost always occurs. Consequently, novel and more effective non-toxic therapies for ovarian cancer are urgently needed. The biosynthetic pathway leading to cholesterol production is an attractive therapeutic target since cholesterol is an essential structural and functional component of cell membranes and is necessary for tumor growth. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, alternative approaches to inhibit cholesterol biosynthesis are being considered. Epithelial ovarian cancer cells express enzymes in the cholesterol biosynthetic pathway. With this is mind we inhibited a key enzyme in the pathway, oxidosqualene cyclase (OSC), with a view to disrupting cholesterol biosynthesis and disrupting the progression of ovarian cancer. Potent small molecule inhibitors of OSC have been identified, and RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating breast and prostate cancer. Since OSC acts downstream of HMG-CoA reductase during cholesterol biosynthesis, its inhibition is not likely to be associated with the adverse effects caused by statins. We found in short-term assays (24-48 h) that pharmacological doses very effectively reduced the viability of ovarian tumor cells (SK-OV-3 and OVCAR-3), determined by Sulforhodamine B colorimetric assay. In long-term assays (7 days), nM concentrations of the drug were also effective. Importantly, RO (20-40 mg/kg/day; ip) significantly suppressed tumor xenografts derived from ovarian cancer cells (SK-OV-3; 336 + 60 mm3 vs 171 + 20 mm3) with no toxicity to experimental animals (27 treatments once tumors reached 100 mm3). Mechanistically, RO induced apoptosis in EOC, and suppressed ALDH activity, indicating that the inhibitor suppresses stem cell-like activity of EOC. Our study is the first to demonstrate disruption of cholesterol biosynthesis in human ovarian cancer cells as a novel and potent means by which to suppress their growth. Further studies will determine whether combining RO with chemotherapeutic drugs is an even more effective therapeutic approach to EOC, and examine the effects of RO on combating the development of drug-resistant ovarian cancer. Presentation: Thursday, June 15, 2023

Abstract IA020: Exploiting cholesterol metabolism for treatment resistant ccRCC patients

Abstract Clear cell renal cell carcinoma (ccRCC) is characterized by large intracellular lipid droplets (LDs) comprised primarily of free and esterified cholesterol molecules; however, the functional significance of cholesterol accumulation in ccRCC cells was unknown. We demonstrated that, surprisingly, genes encoding cholesterol biosynthetic pathway enzymes are strongly repressed in ccRCC cells, making them auxotrophic for exogenous cholesterol. Intriguingly, Mendelian randomization analyses of over 31,000 human genomes indicate a causal link between elevated serum levels of high-density lipoprotein (HDL) cholesterol and ccRCC risk. Depriving ccRCC cells of either cholesterol or HDL compromises proliferation and survival in vitro and ccRCC tumor growth in vivo; in contrast, elevated dietary cholesterol promotes tumor growth. We showed that Scavenger Receptor B1 (SCARB1) is uniquely required for cholesterol import by ccRCC cells, and inhibiting SCARB1 expression or activity is sufficient to cause ccRCC cell cycle arrest and apoptosis accompanied by elevated intracellular reactive oxygen species (ROS) levels and decreased PI3K/AKT signaling. Collectively, our data reveal a previously unrecognized cholesterol dependency in ccRCC and implicate SCARB1 as a therapeutic target that may contribute to novel strategies for treating kidney cancer. Citation Format: M. Celeste Simon. Exploiting cholesterol metabolism for treatment resistant ccRCC patients [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr IA020.

Abstract B073: Inhibiting prostate cancer by targeting the metabolic mevalonate pathway

Abstract Background: A major challenge in the clinical management of prostate cancer (PCa) is inhibiting progression to lethal castrate-resistant PC (CRPC). Deregulated activity of mevalonate (MVA), cholesterol biosynthetic pathway is a recognized hallmark of PCa cells. Statins are potent inhibitors of this metabolic pathway that have been used for decades in the control of hypercholesterolemia. Statins have recently been shown to have anti-PCa activity, however statin treatment triggers a feedback response that restores the MVA pathway, which reduces statin efficacy and contributes to resistance. This restorative feedback loop is controlled by the transcriptional activity of sterol regulatory-element binding protein (SREBP), which primarily induces fatty acid biosynthesis and MVA pathway genes. We have recently identified the anti-platelet agent dipyridamole (DP) as an inhibitor of the statin-induced SREBP-mediated feedback response. However, DP is not SREBP-specific and given its anti-platelet activity, may not be suitable for every cancer patient. Thus, our goal was to identify additional drugs that potentiate the pro-apoptotic activity of statins, which can be used to treat PCa patients. Methods: Two independent, yet complimentary strategies were used. The first focused on performing an in silico analysis to identify drugs that had similar properties to DP at the level of drug structure, molecular perturbations and cell line sensitivity. The second strategy involved a high-content imaging analysis of 1508 FDA approved drugs, which was performed in LNCaP (relatively statin insensitive and feedback competent) and PC3 cells (statin sensitive and feedback incompetent). Cells were treated with a sub-lethal dose of fluvastatin, the drugs or the fluvastatin-drug combination, then treated with apoptotic stains (TMRE, Annexin, Draq 5). Captured images were analyzed using a machine learning approach. Results: Validation of hits from the in silico MVA-DNF approach and high-content screening has identified several drugs that fulfill our criteria of potentiating statin-induced cell death in a feedback-dependent or feedback-independent manner. Interestingly, many show higher Z-scores compared to DP indicating their superior statin potentiation activity to drive PCa cell death. Moreover, a sub-set of these drug significantly inhibit statin-triggered expression of MVA pathway genes HMGCS1 and INSIG1 (p < 0.001) more potently than DP. Conclusions: We have detailed two successful strategies to identify drugs that inhibit SREBP activation in response to statin treatment. These statin-drug combinations represent an effective ‘one-two punch’ to inhibit CRPC progression. These novel inhibitors of SREBP activation potentiate statin at clinically relevant concentrations more potently than DP and have no effect on the platelet activity. Excitingly, many of these agents are FDA-approved and can be immediately used in combination with statins for the treatment of PCa. Overall, our research will lead to novel therapies to improve patient outcome. Citation Format: Diandra Zipinotti dos Santos, Mohamad Elbaz, Emily Branchard, Wiebke Schormann, David W. Andrews, Linda Z. Penn. Inhibiting prostate cancer by targeting the metabolic mevalonate pathway [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B073.

Identifying essential genes in genome-scale metabolic models of consensus molecular subtypes of colorectal cancer.

Identifying essential targets in the genome-scale metabolic networks of cancer cells is a time-consuming process. The present study proposed a fuzzy hierarchical optimization framework for identifying essential genes, metabolites and reactions. On the basis of four objectives, the present study developed a framework for identifying essential targets that lead to cancer cell death and evaluating metabolic flux perturbations in normal cells that have been caused by cancer treatment. Through fuzzy set theory, a multiobjective optimization problem was converted into a trilevel maximizing decision-making (MDM) problem. We applied nested hybrid differential evolution to solve the trilevel MDM problem to identify essential targets in genome-scale metabolic models for five consensus molecular subtypes (CMSs) of colorectal cancer. We used various media to identify essential targets for each CMS and discovered that most targets affected all five CMSs and that some genes were CMS-specific. We obtained experimental data on the lethality of cancer cell lines from the DepMap database to validate the identified essential genes. The results reveal that most of the identified essential genes were compatible with the colorectal cancer cell lines obtained from DepMap and that these genes, with the exception of EBP, LSS, and SLC7A6, could generate a high level of cell death when knocked out. The identified essential genes were mostly involved in cholesterol biosynthesis, nucleotide metabolisms, and the glycerophospholipid biosynthetic pathway. The genes involved in the cholesterol biosynthetic pathway were also revealed to be determinable, if a cholesterol uptake reaction was not induced when the cells were in the culture medium. However, the genes involved in the cholesterol biosynthetic pathway became non-essential if such a reaction was induced. Furthermore, the essential gene CRLS1 was revealed as a medium-independent target for all CMSs.

Characterization of the cholesterol biosynthetic pathway in Dioscorea transversa

Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1β-hydroxyprotoneogracillin and protoneogracillin. Here, we used D.transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D.transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D.transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.

Understanding the molecular mechanisms of statin pleiotropic effects.

Statins represent the cornerstone of pharmacotherapy for the prevention of atherosclerotic cardiovascular disease. These medications not only reduce low-density lipoprotein cholesterol (LDL-C) via inhibition of 3-hydroxy-3-methylglutarate attached to CoA reductase, the key rate-limiting step in the cholesterol biosynthetic pathway, but also upregulate expression of the low-density lipoprotein receptor, improving serum clearance. Given LDL-C is a causal risk factor for the development of atherosclerosis, these complementary mechanisms largely explain why statin therapy leads to reductions in major adverse cardiovascular events. However, decades of basic and clinical research have suggested that statins may exert other effects independent of LDL-C lowering, termed pleiotropic effects, which have become a topic of debate among the scientific community. While some literature suggests statins may improve plaque stability, reduce inflammation and thrombosis, decrease oxidative stress, and improve endothelial function and vascular tone, other studies have suggested potential harmful pleiotropic effects related to increased risk of muscle-related side effects, diabetes, hemorrhagic stroke, and cognitive decline. Furthermore, the introduction of newer, non-statin LDL-C lowering therapies, including ezetimibe, proprotein convertase subtilisin/Kexin Type 9, and bempedoic acid, have challenged the statin pleiotropy theory. This review aims to provide a historical background on the development of statins, explore the mechanistic underpinnings of statin pleiotropy, review the available literature, and provide up to date examples that suggest statins may exert effects outside of LDL-C lowering and the cardiovascular system.

“MiR-7 controls cholesterol biosynthesis through posttranscriptional regulation of DHCR24 expression”

Dysregulation of cholesterol homeostasis is associated with several pathologies including cardiovascular diseases and neurological disorders such as Alzheimer's disease (AD). MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of cholesterol metabolism. We previously established the role of miR-7 in regulating insulin resistance and amyloidosis, which represents a common pathological feature between type 2 diabetes and AD. We show here an additional metabolic function of miR-7 in cholesterol biosynthesis. We found that miR-7 blocks the last steps of the cholesterol biosynthetic pathway in vitro by targeting relevant genes including DHCR24 and SC5D posttranscriptionally. Intracranial infusion of miR-7 on an adeno-associated viral vector reduced the expression of DHCR24 in the brain of wild-type mice, supporting in vivo miR-7 targeting. We also found that cholesterol regulates endogenous levels of miR-7 in vitro, correlating with transcriptional regulation through SREBP2 binding to its promoter region. In parallel to SREBP2 inhibition, the levels of miR-7 and hnRNPK (the host gene of miR-7) were concomitantly reduced in brain in a mouse model of Niemann Pick type C1 disease and in murine fatty liver, which are both characterized by intracellular cholesterol accumulation. Taken together, the results establish a novel regulatory feedback loop by which miR-7 modulates cholesterol homeostasis at the posttranscriptional level, an effect that could be exploited for therapeutic interventions against prevalent human diseases.

Network Analysis Reveals the Molecular Bases of Statin Pleiotropy That Vary with Genetic Background.

Many approved drugs are pleiotropic: for example, statins, whose main cholesterol-lowering activity is complemented by anticancer and prodiabetogenic mechanisms involving poorly characterized genetic interaction networks. We investigated these using the Saccharomyces cerevisiae genetic model, where most genetic interactions known are limited to the statin-sensitive S288C genetic background. We therefore broadened our approach by investigating gene interactions to include two statin-resistant genetic backgrounds: UWOPS87-2421 and Y55. Networks were functionally focused by selection of HMG1 and BTS1 mevalonate pathway genes for detection of genetic interactions. Networks, multilayered by genetic background, were analyzed for key genes using network centrality (degree, betweenness, and closeness), pathway enrichment, functional community modules, and Gene Ontology. Specifically, we found modification genes related to dysregulated endocytosis and autophagic cell death. To translate results to human cells, human orthologues were searched for other drug targets, thus identifying candidates for synergistic anticancer bioactivity. IMPORTANCE Atorvastatin is a highly successful drug prescribed to lower cholesterol and prevent cardiovascular disease in millions of people. Though much of its effect comes from inhibiting a key enzyme in the cholesterol biosynthetic pathway, genes in this pathway interact with genes in other pathways, resulting in 15% of patients suffering painful muscular side effects and 50% having inadequate responses. Such multigenic complexity may be unraveled using gene networks assembled from overlapping pairs of genes that complement each other. We used the unique power of yeast genetics to construct genome-wide networks specific to atorvastatin bioactivity in three genetic backgrounds to represent the genetic variation and varying response to atorvastatin in human individuals. We then used algorithms to identify key genes and their associated FDA-approved drugs in the networks, which resulted in the distinction of drugs that may synergistically enhance the known anticancer activity of atorvastatin.

Comparative Transcriptomic Analysis of Genes in the 20-Hydroxyecdysone Biosynthesis in the Fern Microsorum scolopendria towards Challenges with Foliar Application of Chitosan.

Microsorum scolopendria is an important medicinal plant that belongs to the Polypodiaceae family. In this study, we analyzed the effects of foliar spraying of chitosan on growth promotion and 20-hydroxyecdysone (20E) production in M. scolopendria. Treatment with chitosan at a concentration of 50 mg/L in both young and mature sterile fronds induced the highest increase in the amount of accumulated 20E. Using RNA sequencing, we identified 3552 differentially expressed genes (DEGs) in response to chitosan treatment. The identified DEGs were associated with 236 metabolic pathways. We identified several DEGs involved in the terpenoid and steroid biosynthetic pathways that might be associated with secondary metabolite 20E biosynthesis. Eight upregulated genes involved in cholesterol and phytosterol biosynthetic pathway, five upregulated genes related to the methylerythritol 4-phosphate (MEP) and mevalonate (MVA) pathways, and several DEGs that are members of cytochrome P450s and ABC transporters were identified. Quantitative real-time RT-PCR confirmed the results of RNA-sequencing. Taken together, we showed that chitosan treatment increased plant dry weight and 20E accumulation in M. scolopendria. RNA-sequencing and DEG analyses revealed key enzymes that might be related to the production of the secondary metabolite 20E in M. scolopendria.

Cholesterol Biosynthesis Inhibitor RO 48-8071 Suppresses Growth of Epithelial Ovarian Cancer Cells in Vitro and In Vivo.

Epithelial Ovarian Cancer (EOC) cells express enzymes in the cholesterol biosynthetic pathway, making this pathway an attractive therapeutic target for controlling ovarian cancer. Potent small molecule inhibitors of one biosynthetic enzyme, Oxidosqualene Cyclase (OSC), have been identified, and RO 48-8071 (4'-[6-(allylmethylamino)hexyloxy]-4-bromo-2'-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for breast and prostate cancer. Cell viability assays were performed to determine effects of RO 48-8071 on growth of EOC cells. Aldehyde Dehydrogenase (ALDH) assay was conducted to determine the effects of drug on reducing stem cell like properties of EOC cells. Finally, xenograft studies were performed to assess the ability of RO 48-8071 to inhibit the growth of EOC cells in vivo. We found that short-term (24-48 h) administration of pharmacological doses of RO effectively reduced the viability of drug-resistant EOC cells (SK-OV-3 and OVCAR-3), as determined with sulforhodamine B colorimetric assays. In 7-day assays, nanomolar concentrations of RO effectively inhibited the growth of EOC cells. RO also suppressed ALDH activity, a marker of stem cells. Importantly, RO significantly suppressed growth of xenografts derived from EOC cells when given to mice intraperitoneally (20-40 mg kg-1 day-1) for 27 days once tumors reached 100 mm3 (controls: 336 + 60 mm3; treated: 171 + 20 mm3) with no toxicity to the experimental animals. Mechanistically, RO induced apoptosis in tumor cells in vivo as shown with immunohistochemistry. Cholesterol biosynthesis inhibitor RO 48-8071 is thus a novel and potent inhibitor of human EOC, including EOC stem cells.

Fine-tuning of nitrogen-containing bisphosphonate esters that potently induce degradation of HMG-CoA reductase

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and competitive inhibitors targeting the catalytic domain of this enzyme, so-called statins, are widely used for the treatment of hyperlipidemia. The membrane domain mediates the sterol-accelerated degradation, a post-translational negative feedback mechanism, and small molecules triggering such degradation have been studied as an alternative therapeutic option. Such strategies are expected to provide benefits over catalytic site inhibitors, as the inhibition leads to transcriptional and post-translational upregulation of the enzyme, necessitating a higher dose of the inhibitors and concomitantly increasing the risk of serious adverse effects, including myopathies. Through our previous study on SR12813, a synthetic small molecule that induces degradation of HMG-CoA reductase, we identified a nitrogen-containing bisphosphonate ester SRP3042 as a highly potent HMG-CoA reductase degrader. Here, we performed a systematic structure-activity relationship study to optimize its activity and physicochemical properties, specifically focusing on the reduction of lipophilicity. Mono-fluorination of tert-butyl groups on the molecules was found to increase the HMG-CoA reductase degradation activity while reducing lipophilicity, suggesting the mono-fluorination of saturated alkyl groups as a useful strategy to balance potency and lipophilicity of the lead compounds.

Should oxidosqualene cyclase in the cholesterol biosynthetic pathway be considered an anti-cancer target?

Should oxidosqualene cyclase in the cholesterol biosynthetic pathway be considered an anti-cancer target?