Early life exposure to vitamin D deficiency impairs molecular mechanisms that regulate liver cholesterol biosynthesis, energy metabolism, inflammation, and detoxification.

Introduction: Emerging data suggests liver disease may be initiated during development when there is high genome plasticity and the molecular pathways supporting liver function are being developed. Methods: Here, we leveraged our Collaborative Cross mouse model of developmental vitamin D deficiency (DVD) to investigate the role of DVD in dysregulating the molecular mechanisms underlying liver disease. We defined the effects on the adult liver transcriptome and metabolome and examined the role of epigenetic dysregulation. Given that the parental origin of the genome (POG) influences response to DVD, we used our established POG model [POG1-(CC011xCC001)F1 and POG2-(CC001xCC011)F1] to identify interindividual differences. Results: We found that DVD altered the adult liver transcriptome, primarily downregulating genes controlling liver development, response to injury/infection (detoxification & inflammation), cholesterol biosynthesis, and energy production. In concordance with these transcriptional changes, we found that DVD decreased liver cell membrane-associated lipids (including cholesterol) and pentose phosphate pathway metabolites. Each POG also exhibited distinct responses. POG1 exhibited almost 2X more differentially expressed genes (DEGs) with effects indicative of increased energy utilization. This included upregulation of lipid and amino acid metabolism genes and increased intermediate lipid and amino acid metabolites, increased energy cofactors, and decreased energy substrates. POG2 exhibited broader downregulation of cholesterol biosynthesis genes with a metabolomics profile indicative of decreased energy utilization. Although DVD primarily caused loss of liver DNA methylation for both POGs, only one epimutation was shared, and POG2 had 6.5X more differentially methylated genes. Differential methylation was detected at DEGs regulating developmental processes such as amino acid transport (POG1) and cell growth & differentiation (e.g., Wnt & cadherin signaling, POG2). Conclusions: These findings implicate a novel role for maternal vitamin D in programming essential offspring liver functions that are dysregulated in liver disease. Importantly, impairment of these processes was not rescued by vitamin D treatment at weaning, suggesting these effects require preventative measures. Substantial differences in POG response to DVD demonstrate that the parental genomic context of exposure determines offspring susceptibility.

Squalene-epoxidase-catalyzed 24(S),25-epoxycholesterol synthesis promotes trained-immunity-mediated antitumor activity.

The importance of trained immunity in antitumor immunity has been increasingly recognized, but the underlying metabolic regulation mechanisms remain incompletely understood. In this study, we find that squalene epoxidase (SQLE), a key enzyme in cholesterol synthesis, is required for ß-glucan-induced trained immunity in macrophages and ensuing antitumor activity. Unexpectedly, the shunt pathway, but not the classical cholesterol synthesis pathway, catalyzed by SQLE, is required for trained immunity induction. Specifically, 24(S),25-epoxycholesterol (24(S),25-EC), the shunt pathway metabolite, activates liver X receptor and increases chromatin accessibility to evoke innate immune memory. Meanwhile, SQLE-induced reactive oxygen species accumulation stabilizes hypoxia-inducible factor 1α protein for metabolic switching into glycolysis. Hence, our findings identify 24(S),25-EC as a key metabolite for trained immunity and provide important insights into how SQLE regulates trained-immunity-mediated antitumor activity.

Chemical Inhibition of Sterol Biosynthesis.

Cholesterol is an essential molecule of life, and its synthesis can be inhibited by both genetic and nongenetic mechanisms. Hundreds of chemicals that we are exposed to in our daily lives can alter sterol biosynthesis. These also encompass various classes of FDA-approved medications, including (but not limited to) commonly used antipsychotic, antidepressant, antifungal, and cardiovascular medications. These medications can interfere with various enzymes of the post-lanosterol biosynthetic pathway, giving rise to complex biochemical changes throughout the body. The consequences of these short- and long-term homeostatic disruptions are mostly unknown. We performed a comprehensive review of the literature and built a catalogue of chemical agents capable of inhibiting post-lanosterol biosynthesis. This process identified significant gaps in existing knowledge, which fall into two main areas: mechanisms by which sterol biosynthesis is altered and consequences that arise from the inhibitions of the different steps in the sterol biosynthesis pathway. The outcome of our review also reinforced that sterol inhibition is an often-overlooked mechanism that can result in adverse consequences and that there is a need to develop new safety guidelines for the use of (novel and already approved) medications with sterol biosynthesis inhibiting side effects, especially during pregnancy.

Human oligodendrocyte-like cell differentiation is promoted by TSPO-mediated endogenous steroidogenesis.

Mature oligodendrocytes (OLs) arise from oligodendrocyte precursor cells that, in case of demyelination, are recruited at the lesion site to remyelinate the axons and therefore restore the transmission of nerve impulses. It has been widely documented that exogenously administered steroid molecules are potent inducers of myelination. However, little is known about how neurosteroids produced de novo by OLs can impact this process. Here, we employed a human OL precursor cell line to investigate the role of de novo neurosteroidogenesis in the regulation of OLs differentiation, paying particular attention to the 18 kDa Translocator Protein (TSPO) which controls the rate-limiting step of the neurosteroidogenic process. Our results showed that, over the time of OL maturation, the availability of cholesterol, which is the neurosteroidogenesis initial substrate, and key members of the neurosteroidogenic machinery, including TSPO, were upregulated. In addition, OLs differentiation was impaired following neurosteroidogenesis inhibition and TSPO silencing. On the contrary, TSPO pharmacological stimulation promoted neurosteroidogenic function and positively impacted differentiation. Collectively, our results suggest that de novo neurosteroidogenesis is actively involved in the autocrine and paracrine regulation of human OL differentiation. Moreover, since TSPO was able to promote OL differentiation through a positive modulation of the neurosteroid biosynthetic process, it could be exploited as a promising target to tackle demyelinating diseases.

The constitutively active form of a key cholesterol synthesis enzyme is lipid droplet-localized and upregulated in endometrial cancer tissues.

Cholesterol is essential for both normal cell viability and cancer cell proliferation. Aberrant activity of squalene monooxygenase (SM, also known as squalene epoxidase), the rate-limiting enzyme of the committed cholesterol synthesis pathway, is accordingly implicated in a growing list of cancers. We previously reported that hypoxia triggers the truncation of SM to a constitutively active form, thus preserving sterol synthesis during oxygen shortfalls. Here, we show SM truncation is upregulated and correlates with the magnitude of hypoxia in endometrial cancer tissues, supporting the in vivo relevance of our earlier work. To further investigate the pathophysiological consequences of SM truncation, we examined its lipid droplet-localized pool using complementary immunofluorescence and cell fractionation approaches and found that it exclusively comprises the truncated enzyme. This partitioning is facilitated by the loss of an endoplasmic reticulum-embedded region at the SM N terminus, whereas the catalytic domain containing membrane-associated C-terminal helices is spared. Moreover, we determined multiple amphipathic helices contribute to the lipid droplet localization of truncated SM. Taken together, our results expand on the striking differences between the two forms of SM and suggest upregulated truncation may contribute to SM-related oncogenesis.

7-Dehydrocholesterol dictates ferroptosis sensitivity.

Ferroptosis, a form of regulated cell death that is driven by iron-dependent phospholipid peroxidation, has been implicated in multiple diseases, including cancer1-3, degenerative disorders4 and organ ischaemia-reperfusion injury (IRI)5,6. Here, using genome-wide CRISPR-Cas9 screening, we identified that the enzymes involved in distal cholesterol biosynthesis have pivotal yet opposing roles in regulating ferroptosis through dictating the level of 7-dehydrocholesterol (7-DHC)-an intermediate metabolite of distal cholesterol biosynthesis that is synthesized by sterol C5-desaturase (SC5D) and metabolized by 7-DHC reductase (DHCR7) for cholesterol synthesis. We found that the pathway components, including MSMO1, CYP51A1, EBP and SC5D, function as potential suppressors of ferroptosis, whereas DHCR7 functions as a pro-ferroptotic gene. Mechanistically, 7-DHC dictates ferroptosis surveillance by using the conjugated diene to exert its anti-phospholipid autoxidation function and shields plasma and mitochondria membranes from phospholipid autoxidation. Importantly, blocking the biosynthesis of endogenous 7-DHC by pharmacological targeting of EBP induces ferroptosis and inhibits tumour growth, whereas increasing the 7-DHC level by inhibiting DHCR7 effectively promotes cancer metastasis and attenuates the progression of kidney IRI, supporting a critical function of this axis in vivo. In conclusion, our data reveal a role of 7-DHC as a natural anti-ferroptotic metabolite and suggest that pharmacological manipulation of 7-DHC levels is a promising therapeutic strategy for cancer and IRI.

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

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: 1ß-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.

Augmentation of 3ß-hydroxysteroid-Δ24 Reductase (DHCR24) Expression Induced by Bovine Viral Diarrhea Virus Infection Facilitates Viral Replication via Promoting Cholesterol Synthesis.

Bovine viral diarrhea virus (BVDV) is the etiologic agent of bovine viral diarrhea-mucosal disease, one of the most important viral diseases of cattle, leading to numerous losses to the cattle rearing industry worldwide. The pathogenicity of BVDV is extremely complex, and many underlying mechanisms involved in BVDV-host interactions are poorly understood, especially how BVDV utilizes host metabolism pathway for efficient viral replication and spread. In our previous study, using an integrative analysis of transcriptomics and proteomics, we found that DHCR24 (3ß-hydroxysteroid-Δ24 reductase), a key enzyme in regulating cholesterol synthesis, was significantly upregulated at both gene and protein levels in the BVDV-infected bovine cells, indicating that cholesterol is important for BVDV replication. In the present study, the effects of DHCR24-mediated cholesterol synthesis on BVDV replication was explored. Our results showed that overexpression of the DHCR24 effectively promoted cholesterol synthesis, as well as BVDV replication, while acute cholesterol depletion in the bovine cells by treating cells with methyl-ß-cyclodextrin (MßCD) obviously inhibited BVDV replication. In addition, knockdown of DHCR24 (gene silencing with siRNA targeting DHCR24, siDHCR24) or chemical inhibition (treating bovine cells with U18666A, an inhibitor of DHCR24 activity and cholesterol synthesis) significantly suppressed BVDV replication, whereas supplementation with exogenous cholesterol to the siDHCR24-transfected or U18666A-treated bovine cells remarkably restored viral replication. We further confirmed that BVDV nonstructural protein NS5A contributed to the augmentation of DHCR24 expression. Conclusively, augmentation of the DHCR24 induced by BVDV infection plays an important role in BVDV replication via promoting cholesterol production. IMPORTANCE Bovine viral diarrhea virus (BVDV), an important pathogen of cattle, is the causative agent of bovine viral diarrhea-mucosal disease, which causes extensive economic losses in both cow- and beef-rearing industry worldwide. The molecular interactions between BVDV and its host are extremely complex. In our previous study, we found that an essential host factor 3ß-hydroxysteroid-δ24 reductase (DHCR24), a key enzyme involved in cholesterol synthesis, was significantly upregulated at both gene and protein levels in BVDV-infected bovine cells. Here, we experimentally explored the function of the DHCR24-mediated cholesterol synthesis in regulating BVDV replication. We elucidated that the augmentation of the DHCR24 induced by BVDV infection played a significant role in viral replication via promoting cholesterol synthesis. Our data provide evidence that BVDV utilizes a host metabolism pathway to facilitate its replication and spread.

Caspase-3-Induced Activation of SREBP2 Drives Drug Resistance via Promotion of Cholesterol Biosynthesis in Hepatocellular Carcinoma.

Accumulating evidence has demonstrated that drug resistance can be acquired in cancer through the repopulation of tumors by cancer stem cell (CSC) expansion. Here, we investigated mechanisms driving resistance and CSC repopulation in hepatocellular carcinoma (HCC) as a cancer model using two drug-resistant, patient-derived tumor xenografts that mimicked the development of acquired resistance to sorafenib or lenvatinib treatment observed in patients with HCC. RNA sequencing analysis revealed that cholesterol biosynthesis was most commonly enriched in the drug-resistant xenografts. Comparison of the genetic profiles of CD133+ stem cells and CD133- bulk cells from liver regeneration and HCC mouse models showed that the cholesterol pathway was preferentially upregulated in liver CSCs compared with normal liver stem cells. Consistently, SREBP2-mediated cholesterol biosynthesis was crucial for the augmentation of liver CSCs, and loss of SREBP2 conferred sensitivity to tyrosine kinase inhibitors, suggesting a role in regulation of acquired drug resistance in HCC. Similarly, exogenous cholesterol-treated HCC cells showed enhanced cancer stemness abilities and drug resistance. Mechanistically, caspase-3 (CASP3) mediated cleavage of SREBP2 from the endoplasmic reticulum to promote cholesterol biosynthesis, which consequently caused resistance to sorafenib/lenvatinib treatment by driving activation of the sonic hedgehog signaling pathway. Simvastatin, an FDA-approved cholesterol-lowering drug, not only suppressed HCC tumor growth but also sensitized HCC cells to sorafenib. These findings demonstrate that CSC populations in HCC expand via CASP3-dependent, SREBP2-mediated cholesterol biosynthesis in response to tyrosine kinase inhibitor therapy and that targeting cholesterol biosynthesis can overcome acquired drug resistance. SIGNIFICANCE: This study finds that cholesterol biosynthesis supports the expansion of cancer stem cell populations to drive resistance to tyrosine kinase inhibitor therapy in hepatocellular carcinoma, identifying potential therapeutic approaches for improving cancer treatment.

RNA binding protein RALY activates the cholesterol synthesis pathway through an MTA1 splicing switch in hepatocellular carcinoma.

Alternative splicing is an important RNA processing event that contributes to RNA complexity and protein diversity in cancer. Accumulating evidence demonstrates the essential roles of some alternatively spliced genes in carcinogenesis. However, the potential roles of alternatively spliced genes in hepatocellular carcinoma (HCC) are still largely unknown. Here we showed that the HnRNP Associated with Lethal Yellow Protein Homolog (RALY) gene is upregulated and associated with poor outcomes in HCC patients. RALY acts as a tumor-promoting factor by cooperating with splicing factor 3b subunit 3 (SF3B3) and modulating the splicing switch of Metastasis Associated 1 (MTA1) from MTA-S to MTA1-L. Normally, MTA1-S inhibits cell proliferation by reducing the transcription of cholesterol synthesis genes. In HCC, RALY and SF3B3 cooperate to regulate the MTA1 splicing switch, leading to a reduction in the MTA1-S level, and alleviating the inhibitory effect of MTA1-S on cholesterol synthesis genes, thus promoting HCC cell proliferation. In conclusion, our results revealed that the RALY-SF3B3/MTA1/cholesterol synthesis pathway contributes essentially to hepatic carcinogenesis and could serve as a promising therapeutic target for HCC.

Identification of Four Metabolic Subtypes of Glioma Based on Glycolysis-Cholesterol Synthesis Genes.

Based on alterations in gene expression associated with the production of glycolysis and cholesterol, this research classified glioma into prognostic metabolic subgroups. In this study, data from the CGGA325 and The Cancer Genome Atlas (TCGA) datasets were utilized to extract single nucleotide variants (SNVs), RNA-seq expression data, copy number variation data, short insertions and deletions (InDel) mutation data, and clinical follow-up information from glioma patients. Glioma metabolic subtypes were classified using the ConsensusClusterPlus algorithm. This study determined four metabolic subgroups (glycolytic, cholesterogenic, quiescent, and mixed). Cholesterogenic patients had a higher survival chance. Genome-wide investigation revealed that inappropriate amplification of MYC and TERT was associated with improper cholesterol anabolic metabolism. In glioma metabolic subtypes, the mRNA levels of mitochondrial pyruvate carriers 1 and 2 (MPC1/2) presented deletion and amplification, respectively. Differentially upregulated genes in the glycolysis group were related to pathways, including IL-17, HIF-1, and TNF signaling pathways and carbon metabolism. Downregulated genes in the glycolysis group were enriched in terpenoid backbone biosynthesis, nitrogen metabolism, butanoate metabolism, and fatty acid metabolism pathway. Cox analysis of univariate and multivariate survival showed that risks of glycolysis subtypes were significantly higher than other subtypes. Those results were validated in the CGGA325 dataset. The current findings greatly contribute to a comprehensive understanding of glioma and personalized treatment.

Copy number amplification of ENSA promotes the progression of triple-negative breast cancer via cholesterol biosynthesis.

Copy number alterations (CNAs) are pivotal genetic events in triple-negative breast cancer (TNBC). Here, our integrated copy number and transcriptome analysis of 302 TNBC patients reveals that gene alpha-endosulfine (ENSA) exhibits recurrent amplification at the 1q21.3 region and is highly expressed in TNBC. ENSA promotes tumor growth and indicates poor patient survival in TNBC. Mechanistically, we identify ENSA as an essential regulator of cholesterol biosynthesis in TNBC that upregulates the expression of sterol regulatory element-binding transcription factor 2 (SREBP2), a pivotal transcription factor in cholesterol biosynthesis. We confirm that ENSA can increase the level of p-STAT3 (Tyr705) and activated STAT3 binds to the promoter of SREBP2 to promote its transcription. Furthermore, we reveal the efficacy of STAT3 inhibitor Stattic in TNBC with high ENSA expression. In conclusion, the amplification of ENSA at the 1q21.3 region promotes TNBC progression and indicates sensitivity to STAT3 inhibitors.

Peroxisomal ß-oxidation stimulates cholesterol biosynthesis in the liver in diabetic mice.

Although diabetes normally causes an elevation of cholesterol biosynthesis and induces hypercholesterolemia in animals and human, the mechanism linking diabetes to the dysregulation of cholesterol biosynthesis in the liver is not fully understood. As liver peroxisomal ß-oxidation is induced in the diabetic state and peroxisomal oxidation of fatty acids generates free acetate, we hypothesized that peroxisomal ß-oxidation might play a role in liver cholesterol biosynthesis in diabetes. Here, we used erucic acid, a specific substrate for peroxisomal ß-oxidation, and 10,12-tricosadiynoic acid, a specific inhibitor for peroxisomal ß-oxidation, to specifically induce and suppress peroxisomal ß-oxidation. Our results suggested that induction of peroxisomal ß-oxidation increased liver cholesterol biosynthesis in streptozotocin-induced diabetic mice. We found that excessive oxidation of fatty acids by peroxisomes generated considerable free acetate in the liver, which was used as a precursor for cholesterol biosynthesis. In addition, we show that specific inhibition of peroxisomal ß-oxidation decreased cholesterol biosynthesis by reducing acetate formation in the liver in diabetic mice, demonstrating a crosstalk between peroxisomal ß-oxidation and cholesterol biosynthesis. Based on these results, we propose that induction of peroxisomal ß-oxidation serves as a mechanism for a fatty acid-induced upregulation in cholesterol biosynthesis and also plays a role in diabetes-induced hypercholesterolemia.

Gene and metabolite expression dependence on body mass index in human myocardium.

We hypothesized that body mass index (BMI) dependent changes in myocardial gene expression and energy-related metabolites underlie the biphasic association between BMI and mortality (the obesity paradox) in cardiac surgery. We performed transcriptome profiling and measured a panel of 144 metabolites in 53 and 55, respectively, myocardial biopsies from a cohort of sixty-six adult patients undergoing coronary artery bypass grafting (registration: NCT02908009). The initial analysis identified 239 transcripts with biphasic BMI dependence. 120 displayed u-shape and 119 n-shape expression patterns. The identified local minima or maxima peaked at BMI 28-29. Based on these results and to best fit the WHO classification, we grouped the patients into three groups: BMI < 25, 25 ≤ BMI ≤ 32, and BMI > 32. The analysis indicated that protein translation-related pathways were downregulated in 25 ≤ BMI ≤ 32 compared with BMI < 25 patients. Muscle contraction transcripts were upregulated in 25 ≤ BMI ≤ 32 patients, and cholesterol synthesis and innate immunity transcripts were upregulated in the BMI > 32 group. Transcripts involved in translation, muscle contraction and lipid metabolism also formed distinct correlation networks with biphasic dependence on BMI. Metabolite analysis identified acylcarnitines and ribose-5-phosphate increasing in the BMI > 32 group and α-ketoglutarate increasing in the BMI < 25 group. Molecular differences in the myocardium mirror the biphasic relationship between BMI and mortality.

Gut microbiota promotes cholesterol gallstone formation by modulating bile acid composition and biliary cholesterol secretion.

Cholesterol gallstone disease is a worldwide common disease. Cholesterol supersaturation in gallbladder bile is the prerequisite for its pathogenesis, while the mechanism is not completely understood. In this study, we find enrichment of gut microbiota (especially Desulfovibrionales) in patients with gallstone disease. Fecal transplantation of gut microbiota from gallstone patients to gallstone-resistant strain of mice can induce gallstone formation. Carrying Desulfovibrionales is associated with enhanced cecal secondary bile acids production and increase of bile acid hydrophobicity facilitating intestinal cholesterol absorption. Meanwhile, the metabolic product of Desulfovibrionales, H2S increase and is shown to induce hepatic FXR and inhibit CYP7A1 expression. Mice carrying Desulfovibrionales present induction of hepatic expression of cholesterol transporters Abcg5/g8 to promote biliary secretion of cholesterol as well. Our study demonstrates the role of gut microbiota, Desulfovibrionales, as an environmental regulator contributing to gallstone formation through its influence on bile acid and cholesterol metabolism.

Alteration in glycolytic/cholesterogenic gene expression is associated with bladder cancer prognosis and immune cell infiltration.

BACKGROUND: Oncogenic metabolic reprogramming contributes to tumor growth and immune evasion. The intertumoral metabolic heterogeneity and interaction of distinct metabolic pathways may determine patient outcomes. In this study, we aim to determine the clinical and immunological significance of metabolic subtypes according to the expression levels of genes related to glycolysis and cholesterol-synthesis in bladder cancer (BCa). METHODS: Based on the median expression levels of glycolytic and cholesterogenic genes, patients were stratified into 4 subtypes (mixed, cholesterogenic, glycolytic, and quiescent) in an integrated cohort including TCGA, GSE13507, and IMvigor210. Clinical, genomic, transcriptomic, and tumor microenvironment characteristics were compared between the 4 subtypes. RESULTS: The 4 metabolic subtypes exhibited distinct clinical, molecular, and genomic patterns. Compared to quiescent subtype, mixed subtype was more likely to be basal tumors and was significantly associated with poorer prognosis even after controlling for age, gender, histological grade, clinical stage, and molecular phenotypes. Additionally, mixed tumors harbored a higher frequency of RB1 and LRP1B copy number deletion compared to quiescent tumors (25.7% vs. 12.7 and 27.9% vs. 10.2%, respectively, both adjusted P value< 0.05). Furthermore, aberrant PIK3CA expression level was significantly correlated with those of glycolytic and cholesterogenic genes. The quiescent subtype was associated with lower stemness indices and lower signature scores for gene sets involved in genomic instability, including DNA replication, DNA damage repair, mismatch repair, and homologous recombination genes. Moreover, quiescent tumors exhibited lower expression levels of pyruvate dehydrogenase kinases 1-3 (PDK1-3) than the other subtypes. In addition, distinct immune cell infiltration patterns were observed across the 4 metabolic subtypes, with greater infiltration of M0/M2 macrophages observed in glycolytic and mixed subtypes. However, no significant difference in immunotherapy response was observed across the 4 metabolic subtypes. CONCLUSION: This study proposed a new metabolic subtyping method for BCa based on genes involved in glycolysis and cholesterol synthesis pathways. Our findings may provide novel insight for the development of personalized subtype-specific treatment strategies targeting metabolic vulnerabilities.

Curcumin sensitizes response to cytarabine in acute myeloid leukemia by regulating intestinal microbiota.

PURPOSE: To address whether Curcumin has synergistic effect with cytarabine (Ara-C) in treating acute myeloid leukemia (AML). METHODS: A xenograft AML mouse model was established by injecting HL-60 cells into tail vein of mice to assess the function of Curcumin. Mononuclear cells (MNCs) isolated from AML mice and AML cell lines were used to examine the effect of Curcumin. Metagenomics and metabolomics were used to evaluate the alteration of intestinal microbiota and the change of metabolites in MNCs. RESULTS: Curcumin treatment sensitized response to Ara-C in MNCs of AML mice, but had no direct effect on AML cell lines. Metagenomics revealed an alteration of intestinal microbiota with Curcumin treatment, which contributes to sensitized response to Ara-C. Curcumin treatment led to enhanced intestinal intact to sensitize response to Ara-C in AML mice, through reducing mucus degrading bacteria. Metabolomics demonstrated that Curcumin treatment led to decreased cholesterol in MNCs of AML mice. Further study proved that Curcumin treatment resulted in inhibition of SQLE, a key enzyme of cholesterol biosynthesis, to increase sensitivity to Ara-C. CONCLUSION: Curcumin sensitizes response to Ara-C through regulating microbiota, highlighting the importance of intestinal intact strengthening in chemoresistant therapy. Moreover, aiming at cholesterol synthesis is promising in AML treatment.

RORγ is a context-specific master regulator of cholesterol biosynthesis and an emerging therapeutic target in cancer and autoimmune diseases.

Aberrant cholesterol metabolism and homeostasis in the form of elevated cholesterol biosynthesis and dysregulated efflux and metabolism is well recognized as a major feature of metabolic reprogramming in solid tumors. Recent studies have emphasized on major drivers and regulators such as Myc, mutant p53, SREBP2, LXRs and oncogenic signaling pathways that play crucial roles in tumor cholesterol metabolic reprogramming. Therapeutics such as statins targeting the mevalonate pathway were tried at the clinic without showing consistent benefits to cancer patients. Nuclear receptors are prominent regulators of mammalian metabolism. Their de-regulation often drives tumorigenesis. RORγ and its immune cell-specific isoform RORγt play important functions in control of mammalian metabolism, circadian rhythm and immune responses. Although RORγ, together with its closely related members RORα and RORß were identified initially as orphan receptors, recent studies strongly support the conclusion that specific intermediates and metabolites of cholesterol pathways serve as endogenous ligands of RORγ. More recent studies also reveal a critical role of RORγ in tumorigenesis through major oncogenic pathways including acting a new master-like regulator of tumor cholesterol biosynthesis program. Importantly, an increasing number of RORγ orthosteric and allosteric ligands are being identified that display potent activities in blocking tumor growth and autoimmune disorders in preclinical models. This review summarizes the recent preclinical and clinical progress on RORγ with emphasis on its role in reprogramming tumor cholesterol metabolism and its regulation. It will also discuss RORγ functional mechanisms, context-specificity and its value as a therapeutic target for effective cancer treatment.

Evaluation of lens opacity due to inhibition of cholesterol biosynthesis using rat lens explant cultures.

Drug-induced lens opacity has the potential to cause blindness and is of concern in drug development. Inhibition of cholesterol biosynthesis is one of the causes of lens opacity. Lens opacity is only observed after chronic administration in in vivo nonclinical studies in drug development. Thus, to save resources (e.g., time and cost) and to reduce burden on animals, it is required to develop in vitro evaluation systems that can predict and avoid the risk of lens opacity earlier and easier. In this study, we investigated whether rat lens explant cultures could be useful for the evaluation of drug-induced lens opacity via inhibition of cholesterol biosynthesis. Nineteen drugs, including statins, allylamine, thiocarbamate, azole, and morpholine, which inhibit cholesterol biosynthesis, as well as a negative control (acetaminophen, rosiglitazone and troglitazone), were used. Rat lens explants were treated with drugs for 13 days at concentrations close to IC50 values or higher against cholesterol biosynthesis, and lens opacity (severity and region) was evaluated. In most cases, region-specific lens opacity limited in the equator to posterior pole, as observed in vivo was observed at IC50 values or higher concentrations. The severity of opacity was likely to be related to the inhibitory potency toward cholesterol biosynthesis, concentration of drugs distributed in the lens, or time of exposure. Furthermore, GSH levels were also involved in the deterioration of lens opacity. In conclusion, we demonstrated that rat lens explant cultures can be useful to assess the potential drug-induced lens opacity associated with inhibition of cholesterol biosynthesis and to elucidate the mechanisms of lens opacity.