Fig 1: Restoration of CYP46A1 attenuates the growth of GBM cells in vitro and in intracranial GBM xenografts AqRT–PCR analysis of CYP46A1 mRNA levels in LN229 and GBM#P3 cells transfected with lentivirus expressing CYP46A1 (lenti-CYP46A1) or control sequence (lenti-Ctrl). GAPDH was used as an internal control. Shown are means and SEM (n = 3). LN229: ***P < 0.0001; GBM#P3: ***P < 0.0001. Statistical significance was determined by two-sided Student's t-test.BWestern blot analysis to confirm CYP46A1 overexpression in GBM cells.CGrowth curves for GBM cells in vitro infected with lenti-Ctrl or lenti-CYP46A1 derived from trypan blue staining. Shown are means and SEM (n = 3). LN229: **P = 0.003; GBM#P3: **P = 0.002. Statistical significance was determined by two-sided Student's t-test.DColony-forming assay for GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Shown are means and SEM (n = 3). LN229: *P = 0.013; LN18: *P = 0.012. Statistical significance was determined by two-sided Student's t-test.ERepresentative H&E staining of orthotopic tumours derived from GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Scale bar = 2 mm.FKaplan–Meier survival curve of tumour-bearing mice injected with GBM cells infected with lenti-Ctrl or lenti-CYP46A1 (n = 5 per group). A log-rank test was used to assess statistical significance.GIHC for CYP46A1, PCNA and cleaved caspase-3 protein levels in the intracranial tumours. Scale bar = 100 µm.
Fig 2: Expression of CYP46A1, a gene involved in brain cholesterol homeostasis, is lost in GBM and associated with increasing tumour grade AHeatmap of the differentially expressed cholesterol-related genes between normal brain tissues (n = 28) and glioblastomas (n = 217) from the Rembrandt dataset. Gene expression values are z-transformed and coloured red for high expression and blue for low expression, as indicated in the scale bar.BVolcano plot showing the fold change (log2) in cholesterol-related gene levels based on GBM versus normal brain tissue. Data were obtained from the Rembrandt dataset.CRepresentative images of IHC staining for CYP46A1 protein in normal brain and different pathological grades of gliomas (n = 64). Scale bar = 30 µm.DQuantification of CYP46A1 IHC staining in normal brain (n = 6) and different pathological grades of gliomas (n = 58).ERepresentative images of CYP46A1 IHC staining in GBM and adjacent brain tissues from one specific case. Scale bar = 30 µm.F CYP46A1 expression levels in tumours from the TCGA dataset using 2016 WHO classification. Data are shown as the mean ± the standard error of the mean (SEM; n = 667). ***P < 0.0001. Statistical significance was determined by one-way ANOVA.G CYP46A1 expression levels in different molecular subtypes from the Rembrandt GBM dataset. Shown are means and SEM (n = 245). ***P < 0.0001. Statistical significance was determined by one-way ANOVA.H–IKaplan–Meier analysis for patient OS and PFS based on high versus low expression of CYP46A1 in LGG and GBM. Data were obtained from the CGGA dataset. P-values were obtained from the log-rank test.
Fig 3: CYP46A1 inhibits the growth of GBM cells through the production of 24OHC ASchematic model of cholesterol conversion into 24OHC catalysed by CYP46A1.B24OHC levels in spent media from LN229 (left) and GBM#P3 (right) cells transduced with lenti-Ctrl or lenti-CYP46A1 measured using targeted LC-MS/MS. Data are shown as the mean ± SEM (n = 3). Statistical significance was determined by two-sided Student's t-test (see Materials and Methods).CIC50 curves for 24OHC in LN229 and GBM#P3 cells using the CCK-8 assay.D, EFlow cytometry to detect Annexin V-FITC and PI staining to determine the percentage of LN229 and GBM#P3 cells undergoing apoptosis after exposure to 0–20 µM 24OHC for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: ***P = 0.0005, ***P < 0.0001, ***P < 0.0001; GBM#P3: *P = 0.035, ***P = 0.0006, ***P < 0.0001. Statistical significance was determined by one-way ANOVA.FColony-forming ability of GBM cell lines treated with 24OHC (0–20 µM) for 14 days. Data are shown as the mean ± SEM (n = 3). ***P < 0.0001. Statistical significance was determined by one-way ANOVA.GWestern blot analysis of the apoptosis marker c-PARP, c-caspase-3 and PCNA in lysates (20 µg) from GBM cells treated with 24OHC (0–20 µM) for 72 h.H–ITumorsphere formation assays for GSCs treated with different concentrations of 24OHC (0–20 µM). Scale bar = 100 µm. Graphic representation of the quantification of tumorsphere formation. Data are shown as the mean ± SEM (n = 3). GBM#P3: *P = 0.0192, ***P = 0.0006, ***P = 0.0002; GBM#BG7: **P = 0.0017, ***P < 0.0001, ***P < 0.0001; GBM#BG5: **P = 0.0027, ***P = 0.0001, ***P < 0.0001. Statistical significance was determined by one-way ANOVA.
Fig 4: Expression of CYP46A1 correlates strongly with malignant features in GBM AHeatmap of the differentially expressed cholesterol-related genes between normal brain tissues (n = 5) and glioblastomas (n = 128) from the CGGA dataset. Gene expression values are z-transformed and coloured red for high expression and blue for low expression, as indicated in the scale bar.BVolcano plot showing the fold change (log2) in cholesterol-related gene levels based on GBM versus normal brain tissue samples.CVenn plot showing the significantly dysregulated genes both in Rembrandt and in CGGA datasets.DThe top 20 cholesterol-related genes significantly associated with overall survival in GBM patients from the CGGA dataset.
Fig 5: Efavirenz, an activator of CYP46A1, attenuates the growth of GBM cells and prolongs survival of mice bearing intracranial tumours AThe molecular structure of EFV.BIntracellular concentrations of cholesterol in GBM cells after treatment with 20 µM EFV or DMSO and normalized to total protein. Data are shown as the mean ± SEM (n = 3). LN229: *P = 0.013; GBM#P3: **P = 0.0023. Statistical significance was determined by two-sided Student's t-test.CFilipin staining of GBM cells after treatment with EFV (0–20 µM). Scale bar = 30 µm.DIC50 curves for EFV in GBM cells determined using OD readings (450 nm) from the CCK-8 assay. Data are shown as mean ± SEM (n = 3).E24OHC levels in spent media from LN229 (left) or GBM#P3 (right) cells treated with DMSO or 20 µM EFV for 72 h quantified using targeted LC-MS/MS. Data are shown as the mean ± SEM (n = 3).FImages and quantification of colonies formed by GBM cell lines after treatment with different concentrations of EFV (0–20 µM). Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0056, ***P < 0.0001; LN18: ***P < 0.0001. Statistical significance was determined by one-way ANOVA.GFlow cytometry to detect Annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after treatment with EFV (0–20 µM) for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: ***P = 0.0006, ***P < 0.0001; GBM#P3: ***P < 0.0001. Statistical significance was determined by one-way ANOVA.HTumorsphere formation assays for GSCs treated with different concentrations of EFV (0–20 µM). Scale bar = 100 µm. Graphic representation of the quantification of tumorsphere formation (right). Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0011, ***P = 0.0003, *** P < 0.0001; GBM#BG7: ***P < 0.0001; GBM#BG5: **P = 0.0037, **P = 0.0015, ***P = 0.0002. Statistical significance was determined by one-way ANOVA.IWestern blot analysis of cholesterol homeostasis-related proteins (LDLR, ABCA1, P-SREBP1 and N-SREBP1), c-PARP, PCNA and SOX2 in GBM cells treated with EFV (0–20 µM) for 72 h.JSchematic diagram of the schedule for implantation and drug treatment in the GBM xenograft model. Seven days after implantation of tumour cells, mice were treated with EFV by gavage (0.1 mg/kg/day). Bioluminescent imaging (BLI) using IVIS was performed at days 4, 7, 14 and 21.KThe survival curves of tumour-bearing mice implanted with LN229 or GBM#P3 cells after EFV or PBS treatment (n = 5 per group). A log-rank test was used to assess statistical significance.L, MBioluminescent images and the corresponding quantification of tumours in mice implanted with GBM#P3 cells at days 7 (n = 5 per group, P = 0.58) and 21 (n = 5 per group, P = 0.005). Data are shown as mean ± SEM. Statistical significance was determined by two-sided Student's t-test.NBody weight of tumour-bearing mice after 3 weeks of EFV or PBS treatment. Data are shown as the mean ± SEM (n = 5 per group). Statistical significance was determined by two-sided Student's t-test.
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