Enzyme In Cholesterol Biosynthesis Research Articles

Oleanolic acid inhibits cholesterol synthesis by suppressing the expression and enzymatic activity of HMGCR

ABSTRACT This study investigated the impact of Oleanolic Acid (OA) on cholesterol metabolism and. MTT assays demonstrated that OA had no cytotoxic effects on HepG2 cells at concentrations below 20 μM. Subsequent experiments focused on 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), a key enzyme in cholesterol biosynthesis. OA significantly reduced HMGCR protein expression without affecting other enzymes. Further investigations revealed that OA reduced the protein and mRNA expression of HMGCR and its upstream regulator Sterol regulatory element binding protein-2 (SREBP-2) in HepG2 cells. OA activated Adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK), enhancing HMGCR phosphorylation and inhibiting its activity. Molecular docking experiments suggested that OA could suppress HMGCR enzymatic activity through hydrophobic interactions, reducing catalytic efficiency. Overall, OA downregulated HMGCR expression and activity, leading to decreased cholesterol synthesis. These findings highlight OA’s regulatory role in cholesterol metabolism and its potential for improving atherosclerosis (AS), supporting its application in functional food development.

Abstract 1449: Targeting squalene epoxidase for breast cancer brain metastasis: The evaluation of a new therapeutic target for brain extravasation and colonization using animal models

Abstract Breast cancer brain metastasis is one of the most common forms of breast cancer metastasis and a major cause of morbidity and mortality. Patients who develop brain metastases tend to be associated with progressive neurologic deficits and short overall survival, which represents an unmet medical need. Extensive research has been conducted to elucidate the mechanism of cancer metastasis with limited information gained as to how cancer cells extravasate and colonize to the brain. Recently, we identified a novel mechanism by which squalene epoxidase (SQLE), the second rate-limiting enzyme in cholesterol biosynthesis, plays a critical role in the process of breast cancer metastasis to the brain. Thus, toward a better understanding of the underlying molecular mechanisms and applications of molecular target(s) for brain metastasis therapy, murine models were employed for investigation of brain extravasation and colonization with intracardiac or direct intracranial injections of GFP/FLuc-labeled human MDA-MB-231 metastatic breast carcinoma cells. The MDA-MB-231 cells were found to metastasize to the brain and other organs following intracardiac implantation into nude mice and were further isolated from each tissue. Interestingly, the isolated brain-metastasized tumor cells (MDA-MB-231-BrM) exhibited a significant upregulation of SQLE expression when compared with those that metastasized to other distal sites. The essential role of SQLE in the specific steps of the breast cancer-to-brain metastatic process was evaluated by ex vivo immunofluorescence analysis and immunohistochemistry staining of brain slices from the study subjects. Our data demonstrate that high SQLE expression is essential for MDA-MB-231-BrM to extravasate into the parenchyma, as well as the formation of micro- and macro-metastases in the brain. Interestingly, we found that blood vessel co-option and the surrounding neuronal cells play key roles in supporting MDA-MB-231-BrM to develop brain macro-metastases. In vitro blood-brain barrier (BBB) models further demonstrated the critical role of SQLE in promoting tumor cell invasion and penetration through the BBB. To verify SQLE as an oncogenic factor that can be selected as a potential therapeutic target in suppressing breast cancer brain metastasis, we evaluated the inhibitory effects of terbinafine (a SQLE inhibitor) in both the MDA-MB-231-BrM orthotopic and intracardiac mouse models. Pharmacologic inhibition of SQLE by terbinafine suppressed MDA-MB-231-BrM tumor growth at the mammary fat pad and distal metastases to the brain, suggesting that targeting SQLE represents a therapeutic opportunity for breast cancer brain metastasis. Citation Format: Pony Yu-Ling Lee, Kun-Yuan Lin, Chao-Di Chang, Shan-Yun Cheng, Ya-Wen Hung, Chih-Chieh Yang, Yu-Hsien Chang, Chien-Chang Shen, Jia-Yun Yeh, Yung-Lung Yu, Shu-Ping Wang. Targeting squalene epoxidase for breast cancer brain metastasis: The evaluation of a new therapeutic target for brain extravasation and colonization using animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1449.

Identification and validation of DHCR7 as a diagnostic biomarker involved in the proliferation and mitochondrial function of breast cancer.

Energy metabolism has a complex intersection with pathogenesis and development of breast cancer (BC). This allows for the possibility of identifying energy-metabolism-related genes (EMRGs) as novel prognostic biomarkers for BC. 7-dehydrocholesterol reductase (DHCR7) is a key enzyme of cholesterol biosynthesis involved in many cancers, and in this paper, we investigate the effects of DHCR7 on the proliferation and mitochondrial function of BC. EMRGs were identified from the Gene Expression Omnibus (GEO) and MSigDB databases using bioinformatics methods. Key EMRGs of BC were then identified and validated by functional enrichment analysis, interaction analysis, weighted gene co-expression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO) regression, Cox analysis, and immune infiltration. Western blot, qRT-PCR, immunohistochemistry (IHC), MTT assay, colony formation assay and flow cytometry assay were then used to analyze DHCR7 expression and its biological effects on BC cells. We identified 31 EMRGs in BC. These 31 EMRGs and related transcription factors (TFs), miRNAs, and drugs were enriched in glycerophospholipid metabolism, glycoprotein metabolic process, breast cancer, and cell cycle. Crucially, DHCR7 was a key EMRG in BC identified and validated by WGCNA, LASSO regression and receiver operating characteristic (ROC) curve analysis. High DHCR7 expression was significantly associated with tumor immune infiltration level, pathological M, and poor prognosis in BC. In addition, DHCR7 knockdown inhibited cell proliferation, induced apoptosis and affected mitochondrial function in BC cells. DHCR7 was found to be a key EMRG up-regulated in BC cells. This study is the first to our knowledge to report that DHCR7 acts as an oncogene in BC, which might become a novel therapeutic target for BC patients.

Abstract 5593: METTL3-NUP93 axis as a novel therapeutic target in castration-resistant prostate cancer (CRPC)

Abstract Androgens and androgen receptor (AR) are the key factors driving the development and progression of prostate cancer. Extensive studies confirm both androgens and AR are required for normal growth and development of the prostate gland, and also in prostate cancer pathogenesis and androgen-independent cancer progression. Therefore, androgen deprivation therapies or targeting AR function have always been the mainstay of therapeutic strategy against advanced prostate cancers. However, the current therapeutic strategies are temporally effective in suppressing prostate tumors, and eventually develop metastatic disease. Nuclear pore complex (NPC) is a large complex composed of approximately 30 different proteins termed nucleoporins (NUPs), which serve as the structural and functional units for macromolecules shuttling including proteins and messenger RNAs (mRNAs) between nucleus and cytoplasm. Abnormal expression or genetic alterations of NUP genes has been indicated to involve in the progression of prostate cancer, but the underlying molecular mechanisms are poorly understood. We recently found that the m6A methyltransferase METTL3 directly interacts with nucleoporin 93 (NUP93), one of the NPC components in multiple prostate cancer cells, which seems to be more robust in the castration-resistant prostate cancer (CRPC) cell lines. Disruption of METTL3-NUP93 axis abrogated proliferation and oncogenic phenotypes of CRPC cells, suggesting that the METTL3-NUP93 axis may play an important role in prostate cancer progression to castration resistance. Most importantly, a subgroup of mRNAs regulated through METTL3-NUP93 axis encode cholesterol biosynthesis enzymes that trigger the activation of persistent AR signaling in CRPC. Therefore, Our current findings suggest that inhibition of METTL3-NUP93 axis may represent a promising therapeutic strategy for CRPC, the lethal form of the disease. Citation Format: Jihoon Lee, Zhao Zhang, Juyeong Hong, Kexin Xu. METTL3-NUP93 axis as a novel therapeutic target in castration-resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5593.

Lipid-Restricted Culture Media Reveal Unexpected Cancer Cell Sensitivities.

Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimic the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example, on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provide more realistic levels of lipids. Here, we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. The mechanism of action studies indicates that ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets delta 4-desaturase, sphingolipid 1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies suggest ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and reveal a general screening strategy for identifying additional cancer dependencies masked by the superabundance of medium lipids.

SC5D is the sixth enzyme in cholesterol biosynthesis targeted by the E3 ubiquitin ligase MARCHF6

SC5D is the sixth enzyme in cholesterol biosynthesis targeted by the E3 ubiquitin ligase MARCHF6

Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl β-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

Impact of statins in the liver: A bane or a boon?

Treatment of hypercholesterolemia with statins is considered one of the cornerstones in the management of atherosclerotic cardiovascular diseases. Statins exert their hypolipidemic effects by inhibiting HMG-CoA reductase, the key enzyme in cholesterol biosynthesis. Beyond cholesterol reduction, statins exhibit pleiotropic effects, including anti-inflammatory, antioxidant, and antiproliferative actions, making them valuable in mitigating atherosclerotic and non-atherosclerotic diseases. Though concerns of hepatotoxicity have been associated with the use of statins, extensive evidence suggests that the risk of statin-induced liver injury (SILI) is rare, with an incidence of <1%. Hepatic adverse effects include reversible asymptomatic transaminase elevation (most frequent), hepatitis, cholestasis, and rarely acute liver failure. While hepatotoxicity concerns should not be dismissed, the evidence overwhelmingly supports the safety of statins. Contrary to the myth of statin hepatotoxicity, real-world data and extensive research emphasize the safety and benefits of statins. They are therapeutic in various liver-related conditions, mainly non-alcoholic fatty liver disease. This scientific review aims to provide a comprehensive overview of statins, shedding light on their mechanism of action, hepatotoxicity concerns, and therapeutic potential in various liver-related conditions.

The cholesterol biosynthesis enzyme FAXDC2 couples Wnt/β-catenin to RTK/MAPK signaling.

Wnts, cholesterol, and MAPK signaling are essential for development and adult homeostasis. Here, we report that fatty acid hydroxylase domain containing 2 (FAXDC2), a previously uncharacterized enzyme, functions as a methyl sterol oxidase catalyzing C4 demethylation in the Kandutsch-Russell branch of the cholesterol biosynthesis pathway. FAXDC2, a paralog of MSMO1, regulated the abundance of the specific C4-methyl sterols lophenol and dihydro-T-MAS. Highlighting its clinical relevance, FAXDC2 was repressed in Wnt/β-catenin-high cancer xenografts, in a mouse genetic model of Wnt activation, and in human colorectal cancers. Moreover, in primary human colorectal cancers, the sterol lophenol, regulated by FAXDC2, accumulated in the cancerous tissues and not in adjacent normal tissues. FAXDC2 linked Wnts to RTK/MAPK signaling. Wnt inhibition drove increased recycling of RTKs and activation of the MAPK pathway, and this required FAXDC2. Blocking Wnt signaling in Wnt-high cancers caused both differentiation and senescence; and this was prevented by knockout of FAXDC2. Our data show the integration of 3 ancient pathways, Wnts, cholesterol synthesis, and RTK/MAPK signaling, in cellular proliferation and differentiation.

Shaping immune landscape of colorectal cancer by cholesterol metabolites.

Cancer immunotherapies have achieved unprecedented success in clinic, but they remain largely ineffective in some major types of cancer, such as colorectal cancer with microsatellite stability (MSS CRC). It is therefore important to study tumor microenvironment of resistant cancers for developing new intervention strategies. In this study, we identify a metabolic cue that determines the unique immune landscape of MSS CRC. Through secretion of distal cholesterol precursors, which directly activate RORγt, MSS CRC cells can polarize T cells toward Th17 cells that have well-characterized pro-tumor functions in colorectal cancer. Analysis of large human cancer cohorts revealed an asynchronous pattern of the cholesterol biosynthesis in MSS CRC, which is responsible for the abnormal accumulation of distal cholesterol precursors. Inhibiting the cholesterol biosynthesis enzyme Cyp51, by pharmacological or genetic interventions, reduced the levels of intratumoral distal cholesterol precursors and suppressed tumor progression through a Th17-modulation mechanism in preclinical MSS CRC models. Our study therefore reveals a novel mechanism of cancer-immune interaction and an intervention strategy for the difficult-to-treat MSS CRC.

The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

Transforming growth factor-β1 (TGF-β1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis and hepatocellular carcinoma. However, the reciprocal regulation of TGF-β1 signaling and cholesterol metabolism in MASLD is yet unknown. Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA sequencing of murine hepatocyte cell line (alpha mouse liver 12/AML12) and mouse primary hepatocytes treated with TGF-β1. Functional assays were performed on AML12 cells (untreated, TGF-β1 treated, or subjected to cholesterol enrichment [CE] or cholesterol depletion [CD]), and on mice injected with adenovirus-associated virus 8-control/TGF-β1. TGF-β1 inhibited messenger RNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, mouse primary hepatocytes, and adenovirus-associated virus-TGF-β1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue also were reduced upon TGF-β1 treatment. Smad2/3 phosphorylation after 2 hours of TGF-β1 treatment persisted after CE or CD and was mildly increased after CD, whereas TGF-β1-mediated AKT phosphorylation (30min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by72hours of incubation with TGF-β1, including epithelial-mesenchymal transition, actin polymerization, and apoptosis. CD mimicked the outcome of long-term TGF-β1 administration, an effect that was blocked by an inhibitor of the type I TGF-β receptor. In addition, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. TGF-β1 inhibits cholesterol metabolism whereas cholesterol attenuates TGF-β1 downstream effects in hepatocytes.

10007-ACT-2 FIRST IN HUMAN STUDY OF DSP-0390, AN ORAL EMOPAMIL BINDING PROTEIN (EBP) INHIBITOR, IN RECURRENT HIGH-GRADE GLIOMA (HGG): PRELIMINARY RESULTS

Abstract BACKGROUND Cholesterol in the brain is derived wholly from de novo biosynthesis as the blood-brain barrier prevents uptake from the circulation. The overexpression of cholesterol synthesis genes is associated with decreased survival in glioblastoma (GBM), suggesting that gliomas may be sensitive to the inhibition of cholesterol biosynthesis. DSP-0390 is an investigational small molecule that inhibits EBP, a key enzyme in cholesterol biosynthesis. Preclinical studies demonstrated antitumor activity of DSP-0390 in orthotopic xenograft GBM models. METHODS This phase I dose escalation study aims to assess the safety, PK, PD, and preliminary antitumor activity of oral DSP-0390 monotherapy for recurrent HGG (NCT05023551). Key eligibility criteria include histologically confirmed grade III or IV glioma (WHO 2016); treatment with ≥1 prior therapy; no treatment options beyond standard of care; KPS ≥70. RESULTS As of 9 May 2023, 24 patients (37.5% women) were enrolled across 6 dose levels (20 mg QD to 240 mg QD) with a median age of 51 years (range 24-76) including 17 (70.8 %) with GBM, 3 (12.5%) with anaplastic astrocytoma and 2 (8.3%) with anaplastic oligodendroglioma. Ten patients (41.7%) had IDH mutant tumors. Four patients (16.7%) were on stable dose of corticosteroids at baseline, of which 2 were able to discontinue or dose reduce the corticosteroid while on DSP-0390. There were no reported DLTs. The most common treatment-related TEAEs included grade 1 or 2 nausea (7 patients [29.2%]) and alanine aminotransferase increased (6 patients [25.0%]). Only one grade 3 related AE (dry skin) was noted. Two patients with IDH mutant grade 3 glioma had tumor reduction and remain on DSP-0390 for ≥9 months. Dose dependent changes in PK and PD were observed. CONCLUSIONS DSP-0390 has been well tolerated to date with dose escalation ongoing. A dose response relationship was observed between PK and PD biomarkers.

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.

Abstract C091: Liquiritigenin, an estrogen receptor beta agonist, enhances cholesterol biosynthesis inhibitor RO 48-8071-induced growth inhibition of ovarian cancer cells

Abstract Almost 25,000 new cases of epithelial ovarian cancer (EOC) are reported each year in the United States. Cancers of the ovary have poor prognosis due to late detection, drug resistance and metastasis, and have the highest mortality rate of all the known gynecological malignancies. Despite concerted efforts to develop new strategies for preventing and treating ovarian cancer, novel and more effective non-toxic therapies are urgently needed. Recent observations from our laboratory show that RO 48-8071 ([4’-[6-(Allylmethylamino)hexyloxy]-4-bromo-2’-fluorobenzophenone fumarate] [RO], a small-molecule inhibitor of the key cholesterol biosynthesis enzyme 2, 3-oxidosqualene cyclase (OSC), inhibits breast and prostate cancer cells in vitro and in vivo. Further studies showed that RO also suppresses ovarian cancer cells, both in vitro and in vivo (Cholesterol Biosynthesis Inhibitor RO 48-8071 Suppresses Growth of Epithelial Ovarian Cancer Cells in Vitro and In Vivo. Journal of Cancer Science and Clinical Therapeutics 7 (2023): 01-08.). In short term assays (24h), pharmacological doses of RO (uM) suppressed ovarian cancer cell viability.  In long-term assays (7 days) the same effects were observed in response to RO in the nM range. Interestingly, RO induced the tumor-suppressor protein estrogen receptor-beta (ER-beta) in both breast- and prostate-cancer cells and inhibited growth of xenografts of both types of tumor cells. We tested whether RO treatment also enhanced tumor-suppressor ER-beta expression in ovarian cancer cells and whether ER-beta agonists might enhance the effects of RO.  Using Western blot analysis, we found that RO indeed induced ERb expression in OVCAR-3 ovarian-cancer cells, and that treatment of two ovarian-cancer cell lines (OVCAR-3 and SK-OV-3) with liquiritigenin (LQ), a naturally occurring ER-beta agonist derived from plants, enhanced RO mediated reduction in vitro. Treatment with RO (5-10 uM), LQ (100-300 uM), or RO + LQ (multiple combinations of doses) reduced cell viability; RO + LQ combination treatment synergistically reduced cell viability. We predict that our observed in vitro effects will be translatable to in vivo models, given that we have already demonstrated that RO alone quite effectively inhibits the in vivo growth of EOC xenografts. Since treatment of ovarian cancer with existing anti-cancer drugs is accompanied by extreme side effects and drug resistance, we propose that low-dose combinations of RO and LQ should be further explored as a potential new therapy for combatting this disease. Our findings contribute to ongoing efforts to identify novel, effective treatments for primary and metastatic ovarian cancer that employ new non-toxic drugs. Further in vivo studies examining the effects of RO + LQ combination treatment on EOC are warranted as a means of exploring a potential new therapeutic approach to combat ovarian cancers with minimal toxicity. Citation Format: Yayun Liang, Salman Hyder. Liquiritigenin, an estrogen receptor beta agonist, enhances cholesterol biosynthesis inhibitor RO 48-8071-induced growth inhibition of ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C091.

TRIM28 Regulates DHCR7-Mediated Cholesterol Biosynthesis to Promote Survival in B Cell Lymphoma

Background: TRIM28, a member of the tripartite motif-containing (TRIM) protein family, is a critical regulator of gene expression, DNA damage response and protein degradation, but little is known about its role in lipid metabolism. Dysregulation of cholesterol metabolism has been implicated in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). However, the role of TRIM28 in lymphomagenesis and its clinical relevance remain unclear. Methods: We searched public databases to characterize the gene expression pattern, clinical parameters, and survival data of TRIM28 in B-cell lymphoma, and immunohistochemistry (IHC) was used to confirm TRIM28 expression. Then, we knocked down TRIM28 by lentiviral shRNA transduction in several B-cell lymphoma lines to determine the effect of TRIM28-KD on cell proliferation, cell cycle progression, apoptosis and drug resistance. In addition, a lymphoma xenograft mouse model was established to test whether TRIM28-KD inhibits lymphoma growth in vivo. Using RNA-seq, we aim to identify novel transcriptional targets and elucidate the mechanisms by which TRIM28 promotes survival in B-cell lymphoma. Results: Analysis of bioinformatic datasets and clinical samples indicated that TRIM28 expression was significantly upregulated in B-cell lymphoma compared to normal tissues, and high expression of TRIM28 was closely associated with disease progression and poor prognosis in patients with B-cell lymphoma. Knockdown of TRIM28 inhibited lymphoma cell proliferation, induced cell cycle arrest at G0/G1, increased cell apoptosis, and enhanced the sensitivity of lymphoma cells to chemotherapeutic agents. Moreover, TRIM28-KD significantly suppressed lymphoma and prolonged mouse survival in DLBCL xenograft models. The RNA-seq with TRIM28-KD vs. control revealed differentially expressed genes, many of which were enriched in lipid metabolism-related pathways. DHCR7, a critical terminal enzyme in cholesterol biosynthesis, was identified as a potential transcriptional target of TRIM28. TRIM28-KD inhibited DHCR7 expression, which was associated with reduced total cholesterol levels. DHCR7-KD impaired cell proliferation, whereas overexpression of DHCR7 rescued the phenotypes of TRIM28-KD in lymphoma cells. Conclusions: TRIM28 drives lymphomagenesis through upregulating DHCR7-mediated cholesterol biosynthesis, which may serve as a novel therapeutic target for B-cell lymphoma.

CTNI-67. FIRST IN HUMAN STUDY OF DSP-0390, AN ORAL EMOPAMIL BINDING PROTEIN (EBP) INHIBITOR, IN RECURRENT HIGH-GRADE GLIOMA (HGG): PRELIMINARY RESULTS

Abstract BACKGROUND Cholesterol in the brain is derived wholly from de novo biosynthesis as the blood-brain barrier prevents uptake from the circulation. The overexpression of cholesterol synthesis genes is associated with decreased survival in glioblastoma (GBM), suggesting that gliomas may be sensitive to the inhibition of cholesterol biosynthesis. DSP-0390 is an investigational small molecule that inhibits EBP, a key enzyme in cholesterol biosynthesis. Preclinical studies demonstrated antitumor activity of DSP-0390 in orthotopic xenograft GBM models. METHODS This phase I dose escalation study aims to assess the safety, PK, PD, and preliminary antitumor activity of oral DSP-0390 monotherapy for recurrent HGG (NCT05023551). Key eligibility criteria include histologically confirmed grade III or IV glioma (WHO 2016); treatment with ≥ 1 prior therapy; no treatment options beyond standard of care; KPS ≥ 70. RESULTS As of 9 May 2023, 24 patients (37.5% women) were enrolled across 6 dose levels (20 mg QD to 240 mg QD) with a median age of 51 years (range 24-76) including 17 (70.8 %) with GBM, 3 (12.5%) with anaplastic astrocytoma and 2 (8.3%) with anaplastic oligodendroglioma. Ten patients (41.7%) had IDH mutant tumors. Four patients (16.7%) were on stable dose of corticosteroids at baseline, of which 2 were able to discontinue or dose reduce the corticosteroid while on DSP-0390. There were no reported DLTs. The most common treatment-related TEAEs included grade 1 or 2 nausea (7 patients [29.2%]) and alanine aminotransferase increased (6 patients [25.0%]). Only one grade 3 related AE (dry skin) was noted. Two patients with IDH mutant grade 3 glioma had tumor reduction and remain on DSP-0390 for ≥ 9 months. Dose dependent changes in PK and PD were observed. CONCLUSIONS DSP-0390 has been well tolerated to date with dose escalation ongoing. A dose response relationship was observed between PK and PD biomarkers.

High Plant Protein Diet Ameliorated Hepatic Lipid Accumulation Through the Modulation of Gut Microbiota.

Substituting plant protein for animal protein has emerged as a promising strategy for managing atherogenic lipids. However, the impact of long-term intake of a high plant protein diet (HPD) on hepatic lipid disorder remains unclear. Eight-week-old apolipoprotein E deficient (apoE-/- ) mice are fed with either a normal protein diet (NCD) or HPD for 12 weeks. HPD intervention results in decreased body weight accompanied by increased energy expenditure, with no significant effect on glycemic control. Long-term intake of HPD improves the serum and hepatic lipid and cholesterol accumulation by suppressing hepatic squalene epoxidase (SQLE) expression, a key enzyme in cholesterol biosynthesis. Integrated analysis of 16S rDNA sequencing and metabolomics profiling reveals that HPD intervention increases the abundance of the Lachnospiraece family and serum levels of 12,13-DiHOME. Furthermore, in vivo studies demonstrate that 12,13-DiHOME significantly inhibits lipid accumulation, as well as SQLE expression induced by oleic acid in HepG2 cells. Diet rich in plant protein diet alleviates hyperlipidemia via increased microbial production of 12,13-DiHOME.

A Systematic Review on the Impact of Cholesterol Levels on Diabetes Mellitus

Diabetes mellitus (DM) is a common metabolic disorder characterized by defects in insulin secretion and action, or both, resulting in chronic hyperglycemia and altered metabolism of carbohydrates, fats, and proteins. In the absence of the major risk factors, diabetes is associated with increased morbidity and mortality from cardiovascular disease. The prevalence of diabetes is 10.1 crores, as per the Indian Council of Medical Research – India Diabetes (ICMR INDIAB) study published in 2023. Serum lipid abnormalities (dyslipidemia) are commonly seen in diabetic populations, irrespective of insulin deficiency or insulin resistance. When insulin activity is very low, it severely inhibits lipoprotein lipase production, which significantly impairs the digestion of triglyceride-rich lipoproteins. As a result, there is an increase in triglyceride-rich lipoproteins and a delay in the clearance of chylomicrons and VLDL. Additionally, insulinopenia leads to a substantial increase in lipolysis in adipose tissue, causing the release of free fatty acids into the bloodstream. This higher supply of fatty acids to the liver improves triglyceride synthesis in the liver, ultimately resulting in increased production and secretion of VLDL. By lowering serum cholesterol with statins, which inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, decreases the risk of CHD.

CRYAB plays a role in terminating the presence of pro-inflammatory macrophages in the older, injured mouse peripheral nervous system

Evidence indicates that dysfunction of older Schwann cells and macrophages contributes to poor regeneration of more mature peripheral nervous system (PNS) neurons after damage. Since the underlying molecular factors are largely unknown, we investigated if CRYAB, a small heat shock protein that is expressed by Schwann cells and axons and whose expression declines with age, impacts prominent deficits in the injured, older PNS including down-regulation of cholesterol biosynthesis enzyme genes, Schwann cell dysfunction, and macrophage persistence. Following sciatic nerve transection injury in 3- and 12-month-old wildtype and CRYAB knockout mice, we found by bulk RNA sequencing and RT-PCR, that while gene expression of cholesterol biosynthesis enzymes is markedly dysregulated in the aging, injured PNS, CRYAB is not involved. However, immunohistochemical staining of crushed sciatic nerves revealed that more macrophages of the pro-inflammatory but not immunosuppressive phenotype persisted in damaged 12-month-old knockout nerves. These pro-inflammatory macrophages were more efficient at engulfing myelin debris. CRYAB thus appears to play a role in resolving pro-inflammatory macrophage responses after damage to the older PNS.

Interplay of Cholesterol and Actin in Neurotransmitter GPCR Signaling: Insights from Chronic Cholesterol Depletion Using Statin.

Serotonin1A receptors are important neurotransmitter receptors in the G protein-coupled receptor (GPCR) family and modulate a variety of neurological, behavioral, and cognitive functions. We recently showed that chronic cholesterol depletion by statins, potent inhibitors of HMG-CoA reductase (the rate-limiting enzyme in cholesterol biosynthesis), leads to polymerization of the actin cytoskeleton that alters lateral diffusion of serotonin1A receptors. However, cellular signaling by the serotonin1A receptor under chronic cholesterol depletion remains unexplored. In this work, we explored signaling by the serotonin1A receptor under statin-treated condition. We show that cAMP signaling by the receptor is reduced upon lovastatin treatment due to reduction in cholesterol as well as polymerization of the actin cytoskeleton. To the best of our knowledge, these results constitute the first report describing the effect of chronic cholesterol depletion on the signaling of a G protein-coupled neuronal receptor. An important message arising from these results is that it is prudent to include the contribution of actin polymerization while analyzing changes in membrane protein function due to chronic cholesterol depletion by statins. Notably, our results show that whereas actin polymerization acts as a negative regulator of cAMP signaling, cholesterol could act as a positive modulator. These results assume significance in view of reports highlighting symptoms of anxiety and depression in humans upon statin administration and the role of serotonin1A receptors in anxiety and depression. Overall, these results reveal a novel role of actin polymerization induced by chronic cholesterol depletion in modulating GPCR signaling, which could act as a potential therapeutic target.