The Effects And Mechanisms Of CYP46A1 In The Cholesterol Metabolism Of Glioblastoma

Background:Glioblastoma multiform is the most common primary malignant brain tumor in human adults.Despite aggressive therapy including maximal surgical resection followed by radiotherapy and temozolomide treatment,the median patient survival is 14.6 months from the initial diagnosis.New treatment strategies are therefore urgently needed to improve patients' prognosis.Emerging evidence has linked disrupted cholesterol homeostasis to cancer development including GBM.However,epidemiological data remain contradictory regarding the relationship between cancer risk and serum cholesterol levels,suggesting that circulating cholesterol levels alone have marginal effects on cancer development.Oxysterols are oxygenated derivatives of cholesterol that participate in the regulation of cholesterol metabolism.They are derived from the diet or generated through endogenous cholesterol metabolism in tissues.Oxysterols include 24-hydroxycholesterol(240HC),25-hydroxycholesterol(250HC),27-hydroxycholesterol(270HC),and ring oxysterols that are known to,in addition to cholesterol metabolism regulation,modulate signaling pathways such as Hedgehog,Wnt,and MAPK.In neurodegenerative disease and cancer,oxysterols interact with specific proteins and transcription factors,such as the liver X receptors(LXR)and oxysterol-binding protein,and insulin-induced gene 1(INSIG1).Recently,it has been shown that 270HC can inhibit prostate cancer growth through the depletion of intracellular cholesterol levels.Yet,others have shown,in breast cancer cells,that 270HC induces an epithelial-to-mesenchymal transition(EMT)leading to increased tumor growth.It has also been shown in a subtype of early-stage hepatocellular carcinoma that disrupted cholesterol homeostasis is associated with poor prognosis.In GBM,the notion that excessive accumulation of cholesterol can fuel the growth of tumor cells has been supported by the following evidence.First,the expression of low-density lipoprotein upregulates significantly in GBM,which could uptake a large amount of LDL to acquire cholesterol.Second,loss of endogenous oxysterols in GBM leads to decreased cholesterol efflux.At present,the mechanisms causing dysregulation of cholesterol homeostasis in GBM are not clear,especially with regard to oxysterol loss.In mammals,cholesterol homeostasis is tightly regulated by a complex protein network centered around two groups of transcription factors,sterol-regulatory binding proteins(SREBPs)and liver X receptors(LXRs),that are involved in cholesterol import,export,synthesis,metabolism,and esterification.SREBPs mainly promote the transcription of genes,such as HMGCR and LDLR,that are involved in cholesterol synthesis and uptake when intracellular cholesterol levels decrease.LXRs respond to excessive intracellular cholesterol by inducing the expression of genes,such as ABCA1 and ABCG1,which are responsible for cholesterol efflux and LDLR degradation through induction of the E3 ubiquitin ligase IDOL.Targeting these pathways is an effective strategy for inhibiting growth in GBM animal models.Cancer cells have clearly developed mechanisms for an accumulation of excessive intracellular cholesterol to support their growth.Cholesterol metabolism might therefore represent a potential therapeutic target.However,the key factors contributing to the abnormal accumulation of cholesterol in GBMs remain unclear.Here,we used large-scale in silico analyses of whole-transcriptome databases to identify dysregulated genes in GBMs involved in cholesterol homeostasis.One of the most dramatically down-regulated genes was cholesterol 24-hydroxylase(CYP46A1),a brain-specific enzyme responsible for the elimination of cholesterol through the conversion of cholesterol into 24(S)-hydroxycholesterol(240HC).CYP46A1 expression emerged as a prognostic marker in GBM patients,and in functional studies,overexpression or pharmacological activation of the CYP46A1/240HC axis suppressed GBM cell growth in vitro and in vivo.Our results show that changes in CYP46A1 are critical for the dysregulation of cholesterol homeostasis in GBM and that targeting CYP46A1/240HC may provide a new opportunity for GBM therapy.Part ? Cholesterol promotes the proliferation of GBMObjectives(1)Explore the cholesterol metabolic difference between GBM and normal brain tissues.(2)Reveal the role of cholesterol in GBM proliferation.Methods(1)Analyze the cholesterol metabolism difference between normal brain tissues and glioma tissues by bioinformatic methods.RNA-Seq data of normal brain tissues and glioma specimens in Rembrandt database was extracted to perform GSEA analysis and compare the expression difference of cholesterol metabolism-related genesets.(2)Examine the effects of cholesterol on the proliferation of primary GBM cells and cell lines.A rescue experiment was performed to observe whether the addition of exogenous cholesterol can partially rescue the inhibition of GBM cell proliferation caused by cholesterol lack.Results(1)The way of cholesterol metabolism is different between GBM and normal brain tissues.GSEA showed that cholesterol synthesis-related genesets expression was low in GBM compared to normal brain tissues,which indicated that the proliferation of GBM dependent more on the uptake of exogenous cholesterol.(2)Cholesterol promotes the proliferation of GBM cells.The proliferation of primary GBM cells and GBM cell lines were both inhibited in cholesterol-deficient medium,while addition of LDL(cholesterol-enrichment lipoprotein)could partially rescue the proliferation inhibition resulted from cholesterol deficient.ConclusionsThe cholesterol metabolism is different between normal brain and GBM tissues.Cholesterol is essential for the proliferation of GBM cells.Part ? Cholesterol promotes the proliferation of GBMObjectives(1)Identify the expression of CYP46A1 and its prognostic value in GBM.(2)Reveal the effects of CYP46A1 on the proliferation of GBM.(3)Elucidate the mechanism of CYP46A1 in GBM malignant progression.Methods(1)Identify the most meaningful genes based on integrative analysis of differential expression and prognostic values by extracting cholesterol metabolism-related gene expression and clinicopathological information from Rembrandt and CGGA.Analyze the expression of CYP46A1 in normal brain tissues,gliomas of different grade,and GBM subtypes by retrieving gene expression information in TCGA.Furthermore,verify the prognostic values of CYP46A1 in different databases.(2)IHC was performed to analyze the expression of CYP46A1 in glioma tissues of different WHO grades.Western blot was used to analyze its expression in NHAs and different GBM cells.(3)Construct overexpression lentivirus of CYP46A1 to infect GBM cell lines and primary GBM cells.Examine the overexpression efficiency by q-RT-PCR and Western blot.Evaluate the effects of CYP46A1 overexpression on cell proliferation via cell counting and colony formation assay.(4)Establish an intracranial tumor model of LN229 and GBM#P3 in nude mice to observe the impact of CYP46A1 on the survival of tumor-bearing mice.HE staining is performed to observe the effects of C YP46A1 on tumor volume.(5)Detect the level of 240HC in GBM and normal brain tissues by UHPLC-MS/MS.Detect contents of 240HC after CYP46A1 overexpression.Evaluate the effects of 240HC on the biological behaviors of GBM cells by cell viability assay,cell apoptosis,colony formation,stem cell sphere formation assays,and so on.Western blot was performed to detect relative markers after 240HC stimulation on GBM cells.(6)Examine intracellular cholesterol level after 240HC stimulation by kit and filipin staining.Observe that whether the addition of cholesterol can revert decreased GBM cell malignant behaviors caused by 240HC stimulation.RNA-Seq is used to analyze the effects of 240HC on downstream signals and verify the results by q-RT-PCR and Western blot.Results(1)CYP46A1 is a tumor suppressor gene in GBM.The differential analysis showed that CYP46A1 is one of the most significantly different expression cholesterol metabolism-related genes between normal brain tissues and GBM.Low expression of CYP46A1 predicts a poor prognosis in glioma patients.Public data analysis showed that the expression of CYP46A1 is decreased significantly in GBM and LGG compared to normal brain tissues,which is consistent with IHC results.Western blot indicated that the expression of CYP46A1 decreased in GBM cells compared with NHAs.We observed that abnormal histone modifications resulted in low expression of CYP46A1 in GBM by analyzing ChIP-Seq data from GEO and ENCODE database,which was verified by ChIP assay.(2)Overexpression of CYP46A1 can inhibit GBM cell proliferation.q-RT-PCR and western blot analysis indicate that the expression of CYP46A1 is upregulated in lenti-CYP46A1 infected cells at both mRNA and protein levels.Cell counting,colony formation,and intracranial tumor formation of nude mice show that CYP46A1 can inhibit the proliferation of GBM.Survival analysis implicates that overexpression of CYP46A1 can prolong the survival of tumor-bearing mice.Overexpression of CYP46A1 suppresses the expression of proliferation marker PCNA and increases apoptosis marker cleaved caspase 3 of the intracranial tumor specimen by IHC analysis.(3)CYP46A1 inhibits the proliferation of GBM and GSC stemness by producing 240HC.UHPLC-MS/MS analysis showed that overexpression of CYP46A1 in GBM cells can increase its 240HC contents;it is observed in IC50 assay that 240HC can kill GBM cells significantly in a low concentration.The flow cytometry analysis shows that the apoptosis rate in GBM cells increases significantly by 240HC stimulation.The colony formation and sphere formation ability are decreasing with the elevated level of 240HC concentration.The apoptosis marker is increasing while the proliferation marker is decreasing with the elevated level of 240HC.(4)24OHC inhibits GBM malignant progression via decreasing cholesterol levels.Kit detection and filipin staining show that 240HC stimulation can decrease the cholesterol level of GBM cells.The addition of exogenous cholesterol can partially rescue cell proliferation inhibition and increased the apoptosis rate result from 240HC stimulation.The addition of cholesterol in primary GBM cells can rescue the decreased sphere formation ability caused by 240HC stimulation.(5)240HC inhibits GBM growth by regulating LXR and SREBP1 activity.RNA-Seq showed that those genes downregulated by 240HC are closely related to cholesterol metabolism pathways,which include the target gene of SREBP1 LDLR,a target gene of LXR ABCA1,and markers of glioma stem cells.These results are verified by q-RT-PCR and Western blot.Overexpression of SREBP1 can partially rescue proliferation inhibition caused by 240HC,while SREBP1 inhibitor decreases colony formation ability and intracellular cholesterol levels.These results are similar to those of 240HC stimulation.ConclusionsLoss of CYP46A1 is one of the key factors mediating dysregulated cholesterol homeostasis in glioma.Overexpression of CYP46A1 in GBM cells inhibits its growth by increasing 240HC thus decreasing intracellular cholesterol levels.The underlying mechanism is that CYP46A1 increases 240HC contents,and on one side 240HC can activate LXR thus increasing its downstream gene expression related to cholesterol export,on the other side 240HC inhibits the activation of SREBP1 thus decreasing the expression of its downstream target gene expression like LDLR related to cholesterol uptake.Part ? Efavirenz inhibits glioma proliferation via activating CYP46A1/240HCObjectives(1)Study the therapeutic value of CYP46A1 agonist efavirenz in glioma.(2)Reval the mechanism underlying effects of CYP46A1 agonist efavirenz on inhibiting GBM growth.Methods(1)UHPLC-MS/MS was used to analyze the difference of 240HC contents in primary glioma cells and glioma cell lines after stimulating by CYP46A1 agonist efavirenz.And examine the effects of efavirenz on intracellular cholesterol level of primary glioma cells and glioma cell lines by filipin staining and kits.(2)IC50 assay was used to determine the toxicity of efavirenz on glioma cell lines and primary glioma cells.Colony formation,cell apoptosis,tumorsphere formation assay,and other in vitro assays were performed to examine the effects of efavirenz on GBM cell proliferation,apoptosis,and stemness respectively.Western blot was used to determine the effects of efavirenz on cholesterol metabolism markers,cell apoptosis markers,and tumor stem cell markers.(3)Establish intracranial tumor model of glioma cell line LN229 and primary glioma cell GBM#P3 in nude mice respectively.Record the effects of efavirenz on the survival of tumor-bearing mice and monitor tumor growth by IVIS dynamically.Results(1)Efavirenz can decrease the intracellular cholesterol level of GBM cells.Kit examination shows that intracellular total cholesterol can be decreased by 20 ?M efavirenz stimulation in LN229 and GBM#P3.Besides,filipin staining shows that 10?M and 20?M efavirenz decrease intracellular free cholesterol levels significantly.(2)Efavirenz can kill glioma cells in vitro significantly via increasing 240HC content.IC50 assay indicates that efavirenz has obvious toxic effects on LN229(IC50=16.81?M)and GBM#P3(IC50=24.58 ?M)at low concentrations.240HC in the medium can be elevated significantly by 20?M efavirenz stimulation.Further study implicates that this drug can inhibit colony formation and sphere formation ability of GBM cells as well as increase apoptosis rate,which are accompanied by decreased proliferation marker PCNA and stemness marker SOX2 and increased apoptosis marker c-PARP.On the other side,efavirenz can suppress the expression of cholesterol uptake protein LDLR and transcription factor SREBP1,while increase cholesterol export protein ABCA1.(3)Survival of tumor-bearing mice can be prolonged and tumor growth can be inhibited by efavirenz.In the intracranial GBM tumor model of LN229 and GBM#P3,efavirenz gavage(0.1 mg/kg/day)prolongs median survival of tumor-bearing mice significantly(LN229:n=5,29 vs 35 days,P=0.02;GBM#P3:n=5,27 vs 44 days,P=0.007),and IVIS shows that bioluminescence signal of efavirenz group decreased significantly at day 21(n=5,P=0.005).IHC staining demonstrates that proliferation marker PCNA decreases while apoptosis marker cleaved caspase-3 increases in the efavirenz group.ConclusionsCYP46A1 agonist efavirenz can inhibit GBM cell proliferation by activating CYP46A1 and increase cholesterol metabolite 240HC thus decrease the intracellular cholesterol level.