Cholesterol Pathway Research Articles

Ligusticum chuanxiong prevents high‐fat‐diet‐induced lipid metabolism disorder in mice by modulating the genes in the cholesterol pathway

AbstractLigusticum chuanxiong (LC) has been widely used for cardiovascular and cerebrovascular diseases. LC Hort extraction (LCE) can regulate high‐fat‐diet (HFD)‐induced lipid metabolic disorders (LMDs). However, the potential mechanism of LCE alleviates LMDs has not been entirely determined. This study aimed to investigate the potential effect of LCE in regulating LMDs and reveal its intervention mechanism. LCE was used as the alternative constituent to intervene in HFD‐induced LMDs. LCE antioxidant activity, toxicity, and the effects on serum lipid metabolism index were also determined. The potential intervention mechanism was investigated using the transcriptome analysis. Results confirmed that LCE administration remarkably decreased mice body weight, serum lipid indexes (total cholesterol [TC], triglyceride, low‐density lipoprotein cholesterol, nonesterified fatty acid, and total bile acid), and liver malondialdehyde levels. LCE intervention increased the serum high‐density lipoprotein cholesterol concentration and LPS enzyme activities, and LCE was nontoxic. The liver antioxidantive enzymes, such as catalase, superoxide dismutase, glutathione, lipoprotein lipase, and hepatic lipase, were enhanced. RNA‐seq Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis confirmed that LCE was mainly involved in the lipid metabolism–related signaling pathways, especially for cholesterol biogenesis and metabolic signaling pathways. Besides, genes, such as Cyp51, Msoml, Apof, Pmvk, Nsdh1, ApoA‐1, ApoC‐1, and Lcat, might have been upregulated, thus further inhibiting cholesterol synthesis. By upregulating genes related to the bile acid signaling pathway, such as CYP7A1, CYP27A1, and ABCG5/8, the conversion of TC into bile acid was accelerated, and cholesterol levels decreased. LCE could serve as an alternative Chinese medicine for alleviating HFD‐induced LMDs symptoms through multichannel interactions. This study provides a reference for exploring new functions of LC, especially for regulating LMDs.

BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting.

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

Transcriptome analysis reveals the molecular basis of anti‐predation defence in Daphnia pulex simultaneously responding to Microcystis aeruginosa

Abstract1. The influence of cyanobacteria on interspecies interactions has received growing attention as a predictor of aquatic ecosystem function. It is well‐known that cyanobacteria greatly affect predator‐induced phenotypic plasticity in planktonic animals. However, the underlying molecular mechanism remains unclear.2. Here, we used Daphnia pulex as a model organism for inducible defence, examining its morphological and life‐history responses to fish kairomone while feeding on 100% Chlorella pyrenoides, or while feeding on 70% Chlorella and either microcystin‐producing or ‐free Microcystis aeruginosa, separately. Transcriptome profiles of kairomone‐exposed D. pulex fed different types of food were detected using RNA‐seq. How the differentially expressed genes and pathways identified are responsible for the altered adaptive traits under Microcystis stress was discussed.3. Daphnia pulex fed 100% Chlorella matured at a smaller body size, elongated tail spine length, and produced smaller offspring as adaptive morphological and reproductive responses to fish kairomone. These anti‐predation defences entailed a cost in decreased somatic growth rate. Microcystis stress caused defence trade‐offs in both morphology and reproduction: reduction of body size at maturity was stronger whereas tail spine elongation was unchanged or inhibited. Given that reproductive investment was reduced overall in Daphnia exposed to fish kairomones, there was a trade‐off for unchanged offspring size by reducing offspring number in Daphnia fed Microcystis. Defence‐induced costs to growth were increased by Microcystis exposure.4. Transcriptome analysis revealed that neuronal signals including acetylcholine and glutamate signalling implicated in kairomone reception and/or transmission exhibited stronger responses under Microcystis stress. Cuticle development associated with the biosynthesis of wax esters and chitin, modification of lipo‐chitin saccharides, and the stability of collagen triple helix apparently participated in the morphological changes. The arachidonic acid pathway and biosynthesis of cholesterol and steroid hormones mediated altered reproductive performance. In addition, metabolic processes such as detoxification, food assimilation, lysosome dysfunction and apoptosis activation were involved in the survival and growth of Daphnia, which may contribute to the increased defence‐induced cost in growth when fed Microcystis.5. Taken together, these findings advance our understanding of the molecular mechanisms underlying anti‐predation defensive responses by Daphnia to cyanobacterial stress. This work also provides a reference for further exploring the functional genes that mediate zooplankton–fish coevolution dynamics under eutrophic conditions.

Lanosterol synthase loss of function decreases the malignant phenotypes of HepG2 cells by deactivating the Src/MAPK signaling pathway.

Cholesterol is critical for tumor cells to maintain their membrane components, cell morphology and activity functions. The inhibition of the cholesterol pathway may be an efficient strategy with which to limit tumor growth and the metastatic process. In the present study, lanosterol synthase (LSS) was knocked down by transfecting LSS short hairpin RNA into HepG2 cells, and cell growth, apoptosis and migratory potential were then detected by Cell Counting Kit-8 cell proliferation assay, flow cytometric analysis and wound healing assay, respectively. In addition, proteins associated with the regulation of the aforementioned cell biological behaviors were analyzed by western blot analysis. The activity of the Src/MAPK signaling pathway was measured by western blotting to elucidate the possible signal transduction mechanisms. LSS knockdown in the HepG2 liver cancer cell line inhibited cell proliferation, with cell cycle arrest at the S phase; it also decreased cell migratory ability and increased apoptosis. The expression proteins involved in the regulation of cell cycle, cell apoptosis and migration was altered by LSS knockdown in HepG2 cells. Furthermore, a decreased Src/MAPK activity was observed in the HepG2 cells subjected to LSS knockdown. LSS loss of function decreased the malignant phenotypes of HepG2 cells by deactivating the Src/MAPK signaling pathway and regulating expression of genes involved in cell cycle regulation, cell apoptosis and migration.

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, withkoffrates of P450 51A1-dihydrolanosterol and the 14α-alcohol and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations. epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3β-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-methyl deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14αC-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Oxygen-induced pathological angiogenesis promotes intense lipid synthesis and remodeling in the retina

The retina is a notable tissue with high metabolic needs which relies on specialized vascular networks to protect the neural retina while maintaining constant supplies of oxygen, nutrients, and dietary essential fatty acids. Here we analyzed the lipidome of the mouse retina under healthy and pathological angiogenesis using the oxygen-induced retinopathy model. By matching lipid profiles to changes in mRNA transcriptome, we identified a lipid signature showing that pathological angiogenesis leads to intense lipid remodeling favoring pathways for neutral lipid synthesis, cholesterol import/export, and lipid droplet formation. Noteworthy, it also shows profound changes in pathways for long-chain fatty acid production, vital for retina homeostasis. The net result is accumulation of large quantities of mead acid, a marker of essential fatty acid deficiency, and a potential marker for retinopathy severity. Thus, our lipid signature might contribute to better understand diseases of the retina that lead to vision impairment or blindness.

Abstract 440: Role Of Retinoid X Receptor In Regulating Pcsk9 Transcription In The Liver And Atherosclerosis

Nonalcoholic fatty liver diseases ( NALFD) are associated with high cardiovascular disease ( CVD ) risk. The demand for novel CVD treatments leads to the recent discovery of the Proprotein Convertase Subtilisin Kexin 9 ( PCSK9 ), which promotes the degradation of low-density lipoprotein receptor ( LDLR ) to increase atherogenic lipoprotein LDL cholesterol ( LDL-C ) levels. Targeting PCSK9 shows promising benefits in mitigating CVD and NAFLD. However, the underlying mechanism causing the NAFLD-associated PCSK9 induction is still unclear. Our findings fill the critical knowledge gap by elucidating that Retinoid X Receptor alpha ( RXRα ) is essential to regulate the transcription of PCSK9 in the liver. Our preliminary data showed that hepatic depletion of RXRα leads to hepatic steatosis. To reveal the underlying mechanism by which hepatic RXRα deficiency promotes NAFLD development, we carried out the RNA-seq analysis. The Gene Set Enrichment Analysis ( GSEA ) of RNA-seq data showed the expected downregulation of PPARα signaling genes and the unexpected upregulation of cholesterol pathway genes in the liver of RXRα hepatocyte-specific knockout ( hepKO ) mice. One of the significantly upregulated genes in the cholesterol pathway is PCSK9, which induction in the liver of RXRα hepKO mice can be abolished by AAV8-mediated overexpression of RXRα. Consistently, we observed the increased cholesterol and LDL-C in the plasma of RXRα hepKO mice, and hepatic overexpression of RXRα can significantly reduce the plasma cholesterol and LDL-C levels. Recently, we identified a previously unrecognized acetylation residue of RXRα, which controls the heterodimerization between RXRα and PPARα. The inhibitory effects of constitutively acetylated RXRα mutant further indicate that acetylation of RXRα is essential for preventing the induction of PCSK9 in the liver of RXRα hepKO mice. Interestingly, we also observed the decreased acetylation of RXRα in the liver of high-fat diet ( HFD )-fed mice and human NAFLD patients. These results suggest that RXRα acetylation is required for regulating the PPARα-mediated repression of PCSK9 transcription in the liver, and impaired RXRα acetylation in the NAFLD liver results in increased PCSK9 and atherosclerosis risk.

Abstract 313: Finasteride Delays Atherosclerosis Progression and Reduces Hepatic Inflammation in Low-density Lipoprotein Receptor-deficient Mice

Finasteride is commonly prescribed to treat benign hyperplasia and male-pattern baldness in cis men and, more recently, trans individuals. However, the effect of finasteride on cardiovascular disease remains elusive. We evaluated the role of finasteride on atherosclerosis using low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice fed a Western diet containing increasing doses of finasteride for 12 weeks. We did not observe changes in food intake, body weight, or liver markers for toxicity for any of our experimental conditions. At the highest dose, finasteride reduced plasma cholesterol and triglycerides to approximately 25% compared to the control group. Plaque content at the aortic arch and aortic root level showed a reduction in lesion burden. Finasteride reduced monocytosis, monocyte recruitment to the lesion, macrophage lesion content, and necrotic area. RNA sequencing analysis revealed a downregulation of inflammatory pathways and an upregulation of bile acid metabolism, oxidative phosphorylation, and cholesterol pathways in the liver of mice taking finasteride. Our data unveil finasteride as a potential treatment to delay cardiovascular disease by its effects on plasma lipid profile.

The role of cholesterol metabolism in tumor therapy, from bench to bed.

Cholesterol and its metabolites have important biological functions. Cholesterol is able to maintain the physical properties of cell membrane, play an important role in cellular signaling, and cellular cholesterol levels reflect the dynamic balance between biosynthesis, uptake, efflux and esterification. Cholesterol metabolism participates in bile acid production and steroid hormone biosynthesis. Increasing evidence suggests a strict link between cholesterol homeostasis and tumors. Cholesterol metabolism in tumor cells is reprogrammed to differ significantly from normal cells, and disturbances of cholesterol balance also induce tumorigenesis and progression. Preclinical and clinical studies have shown that controlling cholesterol metabolism suppresses tumor growth, suggesting that targeting cholesterol metabolism may provide new possibilities for tumor therapy. In this review, we summarized the metabolic pathways of cholesterol in normal and tumor cells and reviewed the pre-clinical and clinical progression of novel tumor therapeutic strategy with the drugs targeting different stages of cholesterol metabolism from bench to bedside.

Abstract 5262: Avasimibe abolishes the breast cancer preventative efficacy of statin in a spontaneous mouse model of breast cancer

Abstract Background: The cholesterol biosynthesis pathway plays a central role in the normal cellular development and carcinogenesis. Relevant to breast cancer prevention, the breast epithelium in women with atypical hyperplasia has been shown to have increased in cholesterol levels and oxidative products of cholesterol. Additionally, cholesterol pathway genes such as HMGCR and HMGCS1 are known to be upregulated during progression of breast cancer in patients. We have previously shown in SV40C3TAg mice that the cholesterol lowering drug-fluvastatin reduces breast tumor incidence and burden by 50% and have noted that the efficacy of statin is reduced due to the tight regulation of the cholesterol biosynthesis pathway through multiple restorative feedback loops, thus bypassing the effect of statin blockade. Hypothesis: We hypothesize that fluvastatin efficacy can be improved by co-targeting the restorative feedback pathways that are involved in resistance to statins. Methodology: RNA seq data from statin resistant MCF10.AT1-R cell clones was compared to the statin sensitive parental MCF10.AT1 cells in order to identify pathways and targets involved in statin resistance. Genes that were also found to be upregulated in the statin non-responders mice mammary tumors relative to responders mice were tested for dual targeting. Efficacy of avasimibe, an ACAT inhibitor, to sensitize MCF10.DCIS cells to statin therapy was studied by the colony formation assay. In vivo validation of the statin + avasimibe dual therapy was performed in SV40C3TAg mice that spontaneously develop triple negative breast cancer. Results: We found ACAT1 and ACAT2 to be overexpressed in statin resistance cell clones and non-responder mouse breast tumors. ACATs are involved in cholesterol esterification that is required for storing cholesterol in lipid droplets. Cholesterol esterification in conjunction with cholesterol synthesis and export constitute key mechanisms involved in maintaining cholesterol homeostasis. Thus, we tested if avasimibe treatment potentiates the fluvastatin efficacy to inhibit colonizing ability. Avasimibe and fluvastatin combination completely abolished the ability of MCF10.DCIS cells to form colonies. Next, we tested the efficacy of combination treatment to prevent breast tumors in SV40C3TAg mice. The treatments started at the age of 6 weeks, prior to the onset of cancer and continued until 22 weeks of age. We found fluvastatin and avasimibe combination to be completely ineffective and 90% mice developed tumors. In addition, tumor burden was not reduced with dual treatment. Conclusions: We postulate that avasimibe enhanced metabolism of fluvastatin in mouse system and thus completely abolishing the chemopreventive effects of statin. Genomically derived rationale drug combinations may result in unanticipated interactions and/or ancillary effects that limit efficacy in vivo/in patients. Citation Format: Anjana Bhardwaj, Zhenlin Ju, Alexander Koh, Rhea Bhala, Jing Wang, Isabelle Bedrosian. Avasimibe abolishes the breast cancer preventative efficacy of statin in a spontaneous mouse model of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5262.

Abstract 6122: Targeting mutant p53 and KRAS for novel pancreatic cancer therapy by combination treatment of simvastatin and imipridones

Abstract KRAS mutation is found in 95% pancreatic ductal adenocarcinoma (PDAC). TP53 is altered in 70% of patients with PDAC that co-occur with KRAS mutations. Enhanced de novo cholesterol biosynthesis is a hallmark of cancer cells. P53 inhibits the mevalonate pathway to mediate tumor repression. p53 mutations drive de novo cholesterol pathway activation and are required for the proliferation of KRAS-mutant cancers. Inhibition of the mevalonate pathway leads to feedback activation of SREBP-2, which restores mevalonate pathway and leads to re-activation of KRAS and restoration of mutant p53 GOF. Imipridones induced ATF4 through the action of mitochondria protease ClpP. Simvastatin triggers ATF4 activation through inhibition of mevalonate pathway. We hypothesize that combined treatment with imipridones can overcome the resistance to simvastatin by two different mechanisms: (1) Imipridones inhibit oxidative phosphorylation through ClpP hyperactivation and result in AMPK activation. AMPK phosphorylates and inhibits SREBP-2 transcriptional function, which blocks the feedback activation of SREBP-2. (2) Enhanced ATF4 and CHOP induction through different pathways leads to cancer cell death. We also expect that the combined treatment through sustained induction of ATF4 and CHOP can sensitize cancer cells to imipridone treatment. Mouse and human PDAC cell lines (KPCY, HPAF-II, and PANC-1) with p53 and KRAS mutations were used to test the effects of combination treatment of simvastatin and imipridones. The combined treatments synergistically inhibited the proliferation of p53 and KRAS co-mutant cell lines. For example, the synergy scores of simvastatin and ONC201 combination are 75 (KPCY), 15 (HPAF-II), and 14 (PANC-1). Enhanced cell death is consistent with sustained and enhanced induction of ATF4 and CHOP compared to single drug treatment alone. Imipridones activated AMPK in the tested PDAC cell lines. Imipridones potently inhibit AKT and ERK1/2 activation at 72-hour time point, which might also contribute to block above mentioned feedback activation of KRAS. We also observed that cell lines with a structural mutant of p53 are more sensitive to the combination treatments than a cell line with a DNA-contact mutant of p53, which is consistent with the fact that simvastatin treatment leads to degradation of structural p53 mutants. Surprisingly, our data indicated that simvastatin potently inhibits kinase WEE1 and leads to CDK1 dephosphorylation and activation, which has not been reported before. Together, our data is consistent with publications that p53 mutated cancer cell lines are sensitive to statin treatments. In short, combination treatment may overcome simvastatin resistance and/or imipridone resistance by modulating ATF4/CHOP and SREBP-2 activity. Citation Format: Xiaobing Tian, Wafik S. Deiry. Targeting mutant p53 and KRAS for novel pancreatic cancer therapy by combination treatment of simvastatin and imipridones [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6122.

Biallelic mutations in LSS in autosomal-recessive mutilating palmoplantar keratoderma.

Mutilating palmoplantar keratoderma (PPK) is a heterogeneous genetic disease that poses enormous challenges to clinical diagnosis and genetic counselling. Lanosterol synthase (LSS) gene encodes LSS involved in the biosynthesis pathway of cholesterol. Biallelic mutations in LSS were found to be related to diseases such as cataracts, hypotrichosis and palmoplantar keratoderma-congenital alopecia syndrome. The aim of this study was to investigate the contribution of the LSS mutation to mutilating PPK in a Chinese patient. The clinical and molecular characteristics of the patient were evaluated. A 38-year-old male patient with mutilating PPK was recruited in this study. We identified biallelic variants in the LSS gene (c.683C > T, p.Thr228Ile and c.779G > A, p.Arg260His). Immunoblotting revealed that the Arg260His mutant showed a significantly reduced expression level while Thr228Ile showed an expression level similar to that of the wild type. Thin layer chromatography revealed that mutant Thr228Ile retained partial enzymatic activity and mutant Arg260His did not show any catalytic activity. Our findings show the correlation between LSS mutations and mutilating PPK.

Abstract P6-11-01: Scaffold attachment factor B1 modulates cholesterol pathways in triple-negative breast cancer

Abstract Historically known as a tumor suppressor by estrogen receptor (ER) co-repression in breast cancer (BC), the matrix binding protein Scaffold Attachment Factor B1 (SAFB) binds scaffold or matrix attachment region DNA elements (S/MAR DNA) in eukaryotic DNA. SAFB1 plays a role in cellular stress response, DNA repair, differentiation, and apoptosis. SAFB loss in ER-independent BC and pancreatic cancer (PC) patients resulted in poor survival rates, hinting at the role of SAFB1 as a tumor suppressor. To understand the tumor suppressive mechanism of SAFB1, we performed shRNA-mediated knockdown (KD) of SAFB in PC cell lines and triple-negative breast cancer (TNBC) cells, an aggressive subgroup of BC. Analysis of onco-properties showed an increase in clonogenicity potential and cell proliferation in SAFB KDs in both PDACs and TNBCs cells. Further, RNA-Seq analysis of SAFB KDs in the TNBC cell line revealed activation of the mevalonate (MVA) pathway and the resulting cholesterol biosynthesis as the key metabolic change. Both pancreatic ductal adenocarcinoma (PDACs) and TNBC exhibited higher levels of MVA pathway gene expression upon loss of SAFB. Sterol regulatory element binding proteins (SREBP) 1 and 2 dictate cholesterol biosynthesis, and SREBP2 promotes tumor properties via the MVA pathway. Molecular and metabolic analysis of SAFB KD TNBC showed an increase in lipid droplets and SREBP2 maturation. Using Chromatin Immunoprecipitation and quantitative real-time PCR (ChIP-qPCR) in TNBC we demonstrate the direct binding of SAFB to the SREBP2 promoter region. In addition, byproducts of the MVA pathway have been shown to activate YAP/TAZ-dependent tissue homeostasis and tumorigenesis. We also observed increased YAP1 mRNA levels and decreased YAP1 (Ser127) phosphorylation with SAFB loss in TNBC, explaining the aggressive tumorigenicity gained with SAFB loss. Our study thus far suggests SAFB has overt control over the SREBP2-MVA-YAP1 pathway, and loss of SAFB results in an enhanced tumor phenotype. Citation Format: Meron Ghidey, Divya Murthy, Sukjin Yang, Songyeon Ahn, Jun Hyoung Park, Benny Kaipparettu. Scaffold attachment factor B1 modulates cholesterol pathways in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-01.

Molecular features and predictive models identify the most lethal subtype and a therapeutic target for osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor. The existing treatment regimens remained essentially unchanged over the past 30 years; hence the prognosis has plateaued at a poor level. Precise and personalized therapy is yet to be exploited. One discovery cohort (n=98) and two validation cohorts (n=53 & n=48) were collected from public data sources. We performed a non-negative matrix factorization (NMF) method on the discovery cohort to stratify osteosarcoma. Survival analysis and transcriptomic profiling characterized each subtype. Then, a drug target was screened based on subtypes' features and hazard ratios. We also used specific siRNAs and added a cholesterol pathway inhibitor to osteosarcoma cell lines (U2OS and Saos-2) to verify the target. Moreover, PermFIT and ProMS, two support vector machine (SVM) tools, and the least absolute shrinkage and selection operator (LASSO) method, were employed to establish predictive models. We herein divided osteosarcoma patients into four subtypes (S-I ~ S-IV). Patients of S- I were found probable to live longer. S-II was characterized by the highest immune infiltration. Cancer cells proliferated most in S-III. Notably, S-IV held the most unfavorable outcome and active cholesterol metabolism. SQLE, a rate-limiting enzyme for cholesterol biosynthesis, was identified as a potential drug target for S-IV patients. This finding was further validated in two external independent osteosarcoma cohorts. The function of SQLE to promote proliferation and migration was confirmed by cell phenotypic assays after the specific gene knockdown or addition of terbinafine, an inhibitor of SQLE. We further employed two machine learning tools based on SVM algorithms to develop a subtype diagnostic model and used the LASSO method to establish a 4-gene model for predicting prognosis. These two models were also verified in a validation cohort. The molecular classification enhanced our understanding of osteosarcoma; the novel predicting models served as robust prognostic biomarkers; the therapeutic target SQLE opened a new way for treatment. Our results served as valuable hints for future biological studies and clinical trials of osteosarcoma.

Extra-virgin olive oil ameliorates high-fat diet-induced seminal and testicular disorders by modulating the cholesterol pathway.

Rabbits are sensitive to dietary cholesterol and rapidly develop hypercholesterolemia, leading to prominent subfertility. Sterol regulatory element-binding protein isoform 2 drives the intracellular cholesterol pathway in many tissues, including the testicles. Its abnormal regulation could be the mainly responsible for the failure of suppressing cholesterol synthesis in a cholesterol-enriched environment, ultimately leading to testicular and seminal alterations. However, extra-virgin olive oil consumption has beneficial properties that promote lowering of cholesterol levels, including the recovery of seminal parameters altered under a high-fat diet. Our goal was to investigate the effects of high-fat diet supplementation with extra-virgin olive oil at the molecular level on rabbit testes, by analyzing sterol regulatory element-binding protein isoform 2 protein and its corresponding downstream effectors. During 12months, male rabbits were fed a control diet, high-fat diet, or 6-month high-fat diet followed by 6-month high-fat diet plus extra-virgin olive oil. Serum lipids, testosterone levels, bodyweight, and seminal parameters were tested. The mRNA and protein levels of sterol regulatory element-binding protein isoform 2, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, and low-density lipoprotein receptor were determined by semi-quantitative polymerase chain reaction and Western blotting techniques. The expression pattern of sterol regulatory element-binding protein isoform 2 protein in the rabbit testicles was studied by indirect immunofluorescence. In addition, testicular cholesterol was detected and quantified by filipin staining and gas chromatography. The data showed that the addition of extra-virgin olive oil to high-fat diet reduced testicular cholesterol levels and recovered the expression of sterol regulatory element-binding protein isoform 2, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, and low-density lipoprotein receptor initially altered by the high-fat diet. The combination of high-fat diet with extra-virgin olive oil encourages testicular recovery by modifying the expression of the enzymes related to intracellular cholesterol management.

Role of Lysosomal Cholesterol in Regulating PI(4,5)P2-Dependent Ion Channel Function.

Lysosomes are central regulators of cellular growth and signaling. Once considered the acidic garbage can of the cell, their ever-expanding repertoire of functions include the regulation of cell growth, gene regulation, metabolic signaling, cell migration, and cell death. In this chapter, we detail how another of the lysosome's crucial roles, cholesterol transport, plays a vital role in the control of ion channel function and neuronal excitability through its ability to influence the abundance of the plasma membrane signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This chapter will introduce the biosynthetic pathways of cholesterol and PI(4,5)P2, discuss the molecular mechanisms through which each lipid distinctly regulates ion channels, and consider the interdependence of these lipids in the control of ion channel function.

Transcriptome Profile in the Mouse Brain of Hepatic Encephalopathy and Alzheimer's Disease.

Hepatic encephalopathy (HE) is a chronic metabolic disease accompanied by neuropathological and neuropsychiatric features, including memory deficits, psychomotor dysfunction, depression, and anxiety. Alzheimer's disease (AD), the most common neurodegenerative disease, is characterized by tau hyperphosphorylation, excessive amyloid beta (Aβ) accumulation, the formation of fibrillary tangles, hippocampus atrophy, and neuroinflammation. Recent studies have suggested a positive correlation between HE and AD. Some studies reported that an impaired cholesterol pathway, abnormal bile acid secretion, excessive ammonia level, impaired Aβ clearance, astrocytic dysfunction, and abnormal γ-aminobutyric acid GABAergic neuronal signaling in HE may also be involved in AD pathology. However, the mechanisms and related genes involved in AD-like pathology in the HE brain are unclear. Thus, we compared the cortical transcriptome profile between an HE mouse model, bile duct ligation (BDL), and an AD mouse model, the 5×FAD. Our study showed that the expression of many genes implicated in HE is associated with neuronal dysfunction in AD mice. We found changes in various protein-coding RNAs, implicated in synapses, neurogenesis, neuron projection, neuron differentiation, and neurite outgrowth, and non-coding RNAs possibly associated with neuropathology. Our data provide an important resource for further studies to elucidate AD-like pathophysiology in HE patients.

Long-Term Treatment with Simvastatin Leads to Reduced Migration Capacity of Prostate Cancer Cells.

Statins have been shown to improve survival of metastatic prostate cancer (mPCa). Nevertheless, their therapeutic use is still under debate. In the present study, we investigated the short-term effects of three different statins (simvastatin, atorvastatin and rosuvastatin) in various PCa cell lines mimicking androgen-sensitive and -insensitive PCa. Moreover, we generated three new PCa cell lines (LNCaPsim, ABLsim, PC-3sim) that were cultured with simvastatin over several months. Our data showed that the three statins expressed highly diverse short-term effects, with the strongest growth-inhibitory effect from simvastatin in PC-3 cells and almost no effect from rosuvastatin in any of the cell lines. Long-term treatment with simvastatin resulted in a loss of response to statins in all three cell lines, which was associated with an upregulation of cholesterol and fatty acid pathways as revealed through RNA sequencing. Despite that, long-term treated cells exhibited diminished spheroid growth and significantly reduced migration capacity per se and to differentiated osteoclasts. These findings were strengthened by reduced expression of genes annotated to cell adhesion and migration after long-term simvastatin treatment. Notably, mPCa patients taking statins were found to have lower numbers of circulating tumor cells in their blood with reduced levels of PSA and alkaline phosphatase. Our data suggest that long-term usage of simvastatin hampers the metastatic potential of PCa cells and may therefore be a potential therapeutic drug for mPCa.

Rare Variants in Genes of the Cholesterol Pathway Are Present in 60% of Patients with Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is a pandemic in which conventional risk factors are inadequate to detect who is at risk early in the asymptomatic stage. Although gene variants in genes related to cholesterol, which may increase the risk of AMI, have been identified, no studies have systematically screened the genes involved in this pathway. In this study, we included 105 patients diagnosed with AMI with an elevation of the ST segment (STEMI) and treated with primary percutaneous coronary intervention (PPCI). Using next-generation sequencing, we examined the presence of rare variants in 40 genes proposed to be involved in lipid metabolism and we found that 60% of AMI patients had a rare variant in the genes involved in the cholesterol pathway. Our data show the importance of considering the wide scope of the cholesterol pathway in order to assess the genetic risk related to AMI.

Cholesterol pathway associated with MS disability.

Cholesterol pathway associated with MS disability.