The oncogenic roles of 27-hydroxycholesterol in glioblastoma.

Glioblastoma is the most frequent primary malignant brain tumor in adults. Oxysterols are oxidation products of cholesterol generated by enzymatic reactions. 27-hydroxycholesterol (27-HC), an oxysterol, is an abundant metabolite of cholesterol. 27-HC significantly accelerates mammary cancer growth, proliferation and progression in experimental models. However, to the best of our knowledge, the effect of 27-HC on glioblastoma has not been studied. Therefore, the present study aimed to determine the exact role of 27-HC in glioblastoma. The present study demonstrated that 27-HC promoted proliferation, epithelial to mesenchymal transition, colony formation, migration and invasion of U251 and U118 MG glioblastoma cells. Treatment with 27-HC was also associated with an increase in the formation of glioblastoma-initiating cells in U251 and U118 MG cell lines. Additionally, it was observed that high levels of 27-HC in glioblastoma tissues were associated with poor outcome in patients. In conclusion, 27-HC, a primary metabolite of cholesterol, may serve an important role in the progression of glioblastoma. Copyright: © Liu et al.

Introduction

Glioblastoma is the most frequent primary malignant brain tumor in adults. Median survival is generally <1 year from the time of diagnosis, and even under favorable conditions, the majority of patients succumb to the disease within 2 years (1–3). Consequently, finding novel molecular targets for glioblastoma is critical for improvement of patient outcomes.

27-hydroxycholesterol (27-HC) is an abundant metabolite of cholesterol, which is catalyzed by the P450 enzyme sterol 27-hydroxylase encoded by cytochrome P450 family 27 subfamily A member 1 (4,5). 27-HC is catabolized by another P450 enzyme; cytochrome P450 family 7 subfamily B member 1 (4). 27-HC is an agonist of estrogen receptor α (ERα) action in breast cancer and a competitive antagonist of ERα action in the vasculature (4,6). Notably, it is the first identified endogenous selective ER modulator (5,6). Previous studies have demonstrated that 27-HC significantly accelerates mammary cancer growth, proliferation and progression in experimental models (4,7). In mouse models evaluating MCF-7 ×enografts in ovariectomized animals, 27-HC administration results in increased circulating 27-HC concentrations and increased tumor volume (4,7), whereas growth is halted following 27-HC withdrawal (4). Modulation of 27-HC has been proposed as a potential therapeutic method for breast cancer (4,7), with studies investigating 27-HC antagonism ongoing in breast cancer cell lines (8). However, to the best of our knowledge, the effect of 27-HC has not been studied in glioblastoma. Therefore, the present study aimed to determine the exact role of 27-HC in glioblastoma.

Materials and methods

Patients and tumor samples

Patients with glioblastoma were recruited at the First Affiliated Hospital of Harbin Medical University between July 2016 and December 2016. Information about these patients, including age and sex distribution is shown in Table I. All tissues were examined histologically, and pathologists confirmed the diagnosis. The Medical Ethics Committee of The First Affiliated Hospital of Harbin Medical University approved the experiments undertaken. The use of human tissue samples followed internationally recognized guidelines, as well as local and national regulations. Written informed consent was obtained from each individual. Overall survival was determined from the date of diagnosis to the date of death or last follow-up. The Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis (RPA) classification system for malignant glioma (9,10), previously modified by Shaw et al (11), was used. The modified RTOG RPA classification for glioblastoma considered age, Karnofsky performance status, extent of resection and the ability of patients to perform activities of daily life.

Table I.

Patient characteristics.

Characteristic	Low 27-HC (<194 nM)	High 27-HC (≥194 nM)
Full study population, no. (%)	21 (57)	16 (43)
Age, years
Median (±SD)	59 (±15)	60 (±16)
Sex, no. (%)
Male	14 (68)	9 (56)
Female	7 (32)	7 (44)
RTOG-RPA class, no. (%)
III	4 (17)	3 (16)
IV	13 (62)	10 (65)
V+VI	4 (21)	3 (19)

27-HC, 27-hydroxycholesterol; RTOG-RPA, Radiation Therapy Oncology Group Recursive Partitioning Analysis classification system; SD, standard deviation.

Glioblastoma cell lines

Glioblastoma U251 and U118 MG cell lines were obtained from the Chinese Academy of Sciences Cell Bank and were described previously (12). The cells were authenticated by short tandem repeat profiling. Briefly, cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

Reagents

27-HC and 25-HC were purchased from Yanke, Inc. Cisplatin was obtained from Kangbeibio, Inc. All compounds were solubilized in DMSO (Beyotime Institute of Biotechnology). Following treatment for 48 h, the MTT assay, colony formation assay, western blot analysis, sphere growth assay, and migration and invasion assays were performed as described.

MTT assay

The MTT assay (Beyotime Institute of Biotechnology) was performed as described previously (13). Briefly, cells were plated in 96-well plates in RPMI-1640 medium containing 10% FBS at a density of 8×103 cells/well at 37°C in a 5% CO2 incubator for 12 h. Cells were treated with 27-HC (600 nM), 25-HC (600 nM) or cisplatin (20 µM) for 48 h. Subsequently, MTT (5 mg/ml) was added to the wells (20 µl/well). The plates were incubated at 37°C in 5% CO2 incubator for 4 h; subsequently, the supernatant was removed and 150 µl DMSO was added to each well. After 10 min, the absorbance of each well was measured using a Synergy™ 4 (BioTek Instruments, Inc.) at a wavelength of 570 nm, with the reference wavelength set at 630 nm. Absorbance was directly proportional to the number of live cells.

Colony formation assay

A colony formation assay was performed as described previously (14). Cells untreated or treated with 27-HC or 25-HC (200 cells/well) were seeded in 6-well plates. The culture medium was changed every 2 days. After 14 days of culturing, adherent cells were washed twice with PBS, and fixed with 4% paraformaldehyde for 30 min at room temperature. The colonies were stained with Giemsa solution for 15 min at room temperature and washed with water and air-dried. The colonies were counted using IX70 inverted fluorescence microscope (Olympus Corporation).

Western blot analysis

Western blotting was performed as described previously (15). Total protein was prepared using extraction buffer comprising NaCl/Pi containing 0.5% Triton X-100, 1 mM EDTA, 1 mM phenylmethyl sulfonyl fluoride and 1X complete protease inhibitors (Roche Diagnostics Co., Ltd.). The concentration of each protein lysate was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Total protein (50 µg/lane) were separated by 12% SDS-PAGE. Subsequently, samples were transferred to nitrocellulose membranes and blocked for 60 min at room temperature in 5% skimmed milk powder in NaCl/Pi. The membranes were immunoblotted using the following primary antibodies: Anti-vimentin (cat. no. ab193555; 1:500; Abcam), anti-E-cadherin (cat. no. ab15148; 1:500; Abcam), anti-Stat3 (cat. no. ab76315; 1:500; Abcam), anti-CD133 (cat. no. ab19898; 1:500; Abcam), anti-phosphorylated (p)-MAPK (cat. no. ab185145; 1:500; Abcam), anti-p-mTOR (cat. no. ab2732; 1:500; Abcam), anti-p-AKT (cat. no. ab8805; 1:500 dilution, Abcam), anti-p70S6K (cat. no. ab32529; 1:500; Abcam), anti-YKL40 (cat. no. ab77528; 1:500; Abcam), anti-MAPK (cat. no. ab185145; 1:500; Abcam), anti-mTOR (cat. no. ab2732; 1:500; Abcam), anti-AKT (cat. no. ab18785; 1:500; Abcam), anti-p70S6K (cat. no. ab32529; 1:500; Abcam) and anti-β-actin (cat. no. ab5694; 1:500; Abcam) overnight at 4°C. Subsequently, anti-rabbit secondary antibody (cat. no. ab6940; 1:10,000; Abcam) was used to incubate membranes for 30 min at room temperature. The specific protein bands were visualized by Odyssey™ Infrared Imaging system (LI-COR Biosciences). β-actin expression was used as an internal control to confirm equal loading of the protein samples.

Sphere growth assay

The sphere growth assay was performed as described previously (16). Cells (103/ml) in serum-free RPMI-1640/1 mM Na-pyruvate medium were seeded on 0.5% agar-precoated 6-well plates. After 10 days, half the medium was exchanged every third day. Single spheres were picked and counted. The results were reported as the number of formed spheres per 10,000 cells seeded. Each sphere was imaged using a phase contrast microscope (Leica Microsystems, Inc.) and the diameter was measured using ImageJ software (version 1.47; National Institutes of Health, Bethesda). Spheres with diameters >50 µm were counted.

Migration and invasion assays

Migration and invasion assays were performed as described previously (17). For Transwell migration assays, between 2.5×104 and 5.3×104 cells were plated in the upper chamber with the non-coated membrane (24-well insert; pore size, 8 µm; BD Biosciences). For invasion assays, 1.25×105 cells were plated in the upper chamber with a Matrigel-coated membrane (24-well insert; pore size, 8 µm; BD Biosciences). In the two assays, cells were plated in medium without serum or growth factors in the upper chamber, and medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was used as a chemoattractant in the lower chamber. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h and cells that did not migrate or invade through the pores were removed using a cotton swab. Cells on the lower surface of the membrane were stained with the Diff-Quick Staining set at 37°C for 30 min (Dade Behring; Siemens AG) and counted by a phase contrast microscope (Leica Microsystems, Inc.).

Liquid chromatography-mass spectrometry (LC/MS)

LC/MS was performed as described previously (7,18). Tissues were processed by grinding in liquid nitrogen, followed by the stepwise addition of solvent (H2O); subsequently, 27-HC was extracted from tissues by LC/MS.

Statistical analysis

Statistical analysis was performed with SAS software, version 9.3 (SAS Institute, Inc.). Samples were analyzed by Student's t-test for comparison of two groups (19). If any single group was compared more than once, this counted as multiple comparisons. One-way analysis of variance with Bonferroni multiple-comparisons test was used (19) to compare differences among more than two groups. Overall survival was analyzed by the Kaplan-Meier method (20,21) and log-rank test. Survival was compared in terms of 27-HC concentrations. P<0.05 was considered to indicate a statistically significant difference.

Results

27-HC and 25-hydroxycholesterol (25-HC) promote proliferation and colony formation in U251 and U118 MG cells

To identify the effects of 27-HC and 25-HC on proliferation and colony formation, MTT and colony formation assays were performed on U251 and U118 MG cells. 25-HC weakly promoted proliferation and colony formation of U251 and U118 MG cells (Fig. 1A and B). 27-HC markedly promoted proliferation and colony formation of U251 and U118 MG cells (Fig. 1A and B).

Figure 1.

27-HC and 25-HC promote proliferation and colony formation of U251 and U118 MG glioblastoma cells. (A) MTT assay was conducted to determine the viability of U251 and U118 MG cells. Cells were treated with 27-HC (600 nM), 25-HC (600 nM) and Mock (DMSO). (B) Colony formation assay for U251 cells, performed 48 h after treatment with 27-HC (600 nM), 25-HC (600 nM) and DMSO (Mock). *P<0.05 and **P<0.01 vs. mock group. Error bars indicate the standard error of the mean for experiments performed in triplicate. 25-HC, 25-hydroxycholesterol; 27-HC, 27-hydroxycholesterol; DMSO, dimethyl sulfoxide.

27-HC promotes epithelial to mesenchymal transition (EMT) of U251 and U118 MG cells

To determine whether 27-HC can regulate EMT, U251 and U118 MG cells were treated with 27-HC. Following treatment for 48 h, 27-HC promoted evident alterations in cell morphology (Fig. 2A). During the process, the phenotype changed from a cobblestone-like to a spindle-like morphology (Fig. 2A). To confirm that alterations in morphology were induced by EMT, western blot analysis was carried out to detect epithelial and mesenchymal markers in U251 and U118 MG cells treated with 27-HC or DMSO (Mock). E-cadherin (epithelial marker) expression was inhibited and vimentin (mesenchymal marker) expression was induced by 27-HC in U251 and U118 MG cells (Fig. 2B).

Figure 2.

27-HC promotes epithelial to mesenchymal transition in U251 and U118 MG cells. (A) U251 and U118 MG cells were treated with 27-HC (600 nM) and Mock (DMSO). Images of the cells were then captured after 48-h treatment. (B) Western blot analysis of vimentin and E-cadherin protein expression in U251 and U118 MG cells treated with 27-HC (600 nM) and Mock (DMSO). β-actin was used as a loading control. 25-HC, 25-hydroxycholesterol; 27-HC, 27-hydroxycholesterol; DMSO, dimethyl sulfoxide.

27-HC promotes migration and invasion of U251 and U118 MG cells

Migration and invasion assays were performed in U251 and U118 MG cells treated with 27-HC or DMSO (mock). The present study revealed that 27-HC promoted migration and invasion of glioblastoma cells (Fig. 3).

Figure 3.

27-HC promotes migration and invasion of U251 and U118 MG cells. Migration and invasion assay were conducted on U251 and U118 MG cells. Cells were treated with 27-HC (600 nM) and Mock (DMSO; magnification, ×100). **P<0.01 vs. the mock group. Error bars indicate the standard error of the mean for experiments performed in triplicate. 27-HC, 27-hydroxycholesterol.

27-HC is associated with formation of glioblastoma-initiating cells (GICs)

To determine whether 27-HC could promote formation of GICs, a sphere forming assay was performed to assess formation of a stem cell-like population. The formation of spheres was increased in U251 and U118 MG cells treated with 27-HC (Fig. 4A). In order to detect whether expression of Stat3, a documented marker for GICs (22), may be affected by 27-HC, western blot analysis was carried out. The results revealed that Stat3 expression was upregulated by 27-HC in U251 and U118 MG cells (Fig. 4B).

Figure 4.

27-HC is associated with increased formation of glioblastoma-initiating cells. (A) Sphere growth assay for U251 and U118 MG cells (magnification, ×100). Cells were treated with 27-HC (600 nM) and Mock (DMSO). (B) Western blot analysis of Stat3 protein expression in U251 and U118 MG cells treated with 27-HC and Mock (DMSO). **P<0.01 vs. mock group. β-actin was used as a loading control. Error bars indicate the standard error of the mean for experiments performed in triplicate. 27-HC, 27-hydroxycholesterol; DMSO, dimethyl sulfoxide; Stat3, signal transducer and activator of transcription 3.

27-HC is a prognostic biomarker for patients with glioblastoma

LC/MS was performed to determine the concentration of 27-HC in glioblastoma. Concentrations <194 nM were defined as low levels of 27-HC and concentrations ≥194 nM were defined as high levels of 27-HC. Kaplan-Meier curves were applied to assess overall survival for 37 human patients with glioblastoma, stratified based on concentration of tumor 27-HC. Using a log-rank test, it was determined that the two overall survival curves were significantly different (Fig. 5). Patients with high tumor 27-HC concentrations exhibited much poorer overall survival compared with patients with low 27-HC concentrations (Fig. 5).

Figure 5.

27-HC is associated with shorter overall survival of patients with glioblastoma. Kaplan-Meier curves for overall survival of 37 patients with glioblastoma, stratified based on the concentration of tumor 27-HC. Each drop in the survival curve represents the time of ≥1 deaths. The blips represent the time of censoring (the ‘blips’ represent the crosses in the low concentration curve). The P-value was calculated using a log-rank test. **P<0.01 vs. mock group. 27-HC, 27-hydroxycholesterol.

27-HC promotes cisplatin resistance of U251 and U118 MG cells

To identify whether 27-HC could affect cisplatin efficacy, an MTT assay was performed in U251 and U118 MG cells treated as indicated (Fig. 6A). 27-HC promoted cisplatin resistance (Fig. 6A). Western blot analysis was used to determine the expression of CD133 in U251 and U118 MG cells. The results revealed that the expression of CD133, a documented marker for cisplatin resistance (23), was upregulated by 27-HC in U251 and U118 MG cells (Fig. 6B).

Figure 6.

27-HC promotes cisplatin resistance of U251 and U118 MG cells. (A) MTT assay was used to detect the viability of U251 and U118 MG cells. Cells treated with 27-HC (600 nM) or Mock (DMSO) were treated with or without cisplatin (20 µM). (B) Western blot analysis of CD133 protein expression in U251 and U118 MG cells treated with 27-HC (600 nM) and Mock (DMSO). β-actin was used as a loading control. *P<0.05 and **P<0.01 vs. mock group. Error bars indicate the standard error of the mean for experiments performed in triplicate. 27-HC, 27-hydroxycholesterol; CD133, cluster of differentiation 133; DMSO, dimethyl sulfoxide.

27-HC regulates p-MAPK, p-mTOR, p-AKT, p-p70S6K and YKL40 expression in U251 and U118 MG cells. To identify whether 27-HC could regulate MAPK, p-MAPK, mTOR, p-mTOR, AKT, p-AKT, p70S6K, p-p70S6K and YKL40 expression, western blot analysis was performed for U251 and U118 MG cells treated as indicated (Fig. 7). The results revealed that 27-HC promoted p-MAPK, p-mTOR, p-AKT, p-p70S6K and YKL40 expression (Fig. 7A). However, the expression levels of MAPK, mTOR, AKT and p70S6K were not affected by the treatment with 27-HC in U251 and U118 MG cells (Fig. 7B).

Figure 7.

27-HC regulates p-MAPK, p-mTOR, p-Akt, p-p70S6K and YKL40 expression in U251 and U118 MG cells. (A) Western blot analysis of p-MAPK, p-mTOR, p-Akt, p-p70S6K and YKL40 protein expression in U251 and U118 MG cells treated with 27-HC (600 nM) and Mock (DMSO). (B) Western blot analysis of MAPK, mTOR, Akt and p70S6K protein expression in U251 and U118 MG cells treated with 27-HC (600 nM) and Mock (DMSO). β-actin was used as a loading control. 27-HC, 27-hydroxycholesterol; Akt, protein kinase B; DMSO, dimethyl sulfoxide; MAPK, mitogen-activated protein kinase; mTOR, mechanistic target of rapamycin; p-, phosphorylated; p70S6K, ribosomal protein S6 kinase β-1; YKL40, chitinase-3-like protein 1.

Discussion

The present study, to the best of our knowledge, was the first to evaluate the roles of 27-HC and 25-HC in glioblastoma. The results of the present study suggested that 27-HC, a primary metabolite of cholesterol, may have important functions in the progression of glioblastoma. Additionally, 25-HC weakly promoted proliferation and colony formation in glioblastoma cells.

Transitions between epithelial and mesenchymal states lead to glioblastoma progression (24,25). EMT is a key process contributing to glioblastoma metastasis and poor prognosis, characterized by a decrease in the expression of epithelial markers (e.g. E-cadherin) and an increase in mesenchymal markers (e.g. vimentin) (26). During the process of EMT, a phenotypic alteration from a cobblestone-like to a spindle-like morphology is one of the major hallmarks of malignant transformation of glioblastoma cells (24,25,27). In the present study, 27-HC caused significant changes in the morphology of glioblastoma cells (phenotypic alteration from a cobblestone-like to a spindle-like morphology). Additionally, E-cadherin expression was inhibited and vimentin expression was induced by the administration of 27-HC in glioblastoma cells. These findings suggested that 27-HC promoted EMT by regulating E-cadherin and vimentin expression in glioblastoma cells.

EMT serves an important role in the formation of GICs (28). In the present study, 27-HC promoted the formation of GICs. It has been reported that increased GIC formation promotes migration and invasion of glioblastoma (29). Consistent with the previous study (29), 27-HC promoted migration and invasion of glioblastoma cells in the present study. In addition, increased GIC formation has been associated with enhanced colony formation in numerous types of cancer (30). In the present study, 27-HC promoted colony formation in human glioblastoma cells.

Chemoresistance is a major obstacle for the treatment of glioblastoma (31). A number of different mechanisms may account for this chemoresistance, including upregulation of anti-apoptotic pathways, enhanced DNA repair and the existence of GICs (32,33). In the present study, 27-HC promoted cisplatin resistance in U251 and U118 cells. Increased p-MAPK, p-mTOR, p-AKT, p-p70S6K and YKL40 expression levels have been associated with poor prognosis of patients with glioblastoma (34). In the present study, 27-HC promoted p-MAPK, p-mTOR, p-AKT, p-p70S6K and YKL40 expression in glioblastoma cells. In addition, high 27-HC concentration was associated with poor patient outcome, which is in line with these experimental results.

The U118 MG cell line has been identified as a derivative of U138 MG cells (35). However, the U118 MG cell line is still widely used for glioblastoma research (36). In the present study, U251 and U118 MG cells were used. The results were similar for the two cell lines.

In conclusion, the present study provided molecular and clinical implications for the role of 27-HC in glioblastoma. 27-HC may be an oncogenic metabolite of cholesterol and a potential driver of disease progression in glioblastoma. In addition, higher 27-HC expression levels were associated with shorter overall survival; therefore, 27-HC may be used as a prognostic biomarker of glioblastoma.