Alpha-synuclein contributes to malignant progression of human meningioma via the Akt/mTOR pathway
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The aim of this study is to explore the expression of alpha-synuclein (α-synuclein) in benign, atypical, and anaplastic meningiomas and determine its role in the malignant progression of meningiomas.
Expression of α-synuclein was measured in 44 meningioma samples by real-time PCR analysis. The effects of overexpression or knockdown of α-synuclein on meningioma cell growth, invasiveness, and tumorigenicity were determined.
Atypical and anaplastic meningiomas displayed significantly greater levels of α-synuclein mRNA, relative to benign tumors. Depletion of α-synuclein decreased cell proliferation and colony formation and promoted apoptosis in IOMM-Lee meningioma cells, whereas overexpression of α-synuclein facilitated cell proliferation and colony formation in CH-157MN meningioma cells. Silencing of α-synuclein attenuated IOMM-Lee cell migration and invasion. In contrast, ectopic expression of α-synuclein increased the invasiveness of CH-157MN cells. In vivo studies further demonstrated that downregulation of α-synuclein significantly retarded meningioma growth in nude mice. At the molecular level, the phosphorylation levels of Akt, mTOR, p70S6K and 4EBP were significantly decreased in α-synuclein-depleted IOMM-Lee cells.
In conclusion, α-synuclein upregulation contributes to aggressive phenotypes of meningiomas via the Akt/mTOR pathway and thus represents a potential therapeutic target for malignant meningiomas.
KeywordsAkt/mTOR signaling α-Synuclein Growth Invasion Meningioma
Dulbecco’s modified Eagle medium
fetal bovine serum
quantitative real-time PCR
small hairpin RNA
Meningiomas are mesenchymal tumors that account for ~30% of all primary intracranial tumors . Most meningiomas are benign tumors of World Health Organization (WHO) grade I. However, up to 20% of meningiomas have aggressive behaviors, which are classified as atypical WHO grade II or anaplastic WHO grade III . The Akt/mTOR pathway has been suggested to be involved in the pathogenesis of meningiomas [3, 4]. However, the molecular mechanisms underlying the malignant progression of meningiomas are still elusive.
Alpha-synuclein (α-synuclein) is a soluble presynaptic protein belonging to the synuclein protein family. It is well known that α-synuclein can form insoluble fibril aggregates and play a pathogenic role in Parkinson’s disease (PD) . In addition to expression in nervous tissues, α-synuclein is expressed in other tissue types such as melanomas [6, 7, 8]. It was found that α-synuclein is abundantly presented in primary and metastatic melanomas, but undetectable in non-melanocytic cutaneous carcinoma and normal skin . There is evidence that α-synuclein differentially regulates the synthesis of melanin in melanoma and dopaminergic neuronal cells, consequently affecting cell susceptibility to ultra-violet radiation-induced injury . A previous study has demonstrated that α-synuclein is expressed in a variety of brain tumors, but not in benign meningiomas . However, the expression and biological relevance of α-synuclein in the malignant progression of meningiomas are not clarified.
In this study, we compared the expression of α-synuclein in atypical and anaplastic meningiomas versus benign tumors and explored the roles of α-synuclein in regulating the aggressive phenotypes of meningiomas.
A total of 44 meningioma specimens (18 benign, 17 atypical, and 9 anaplastic meningiomas) were obtained from patients who underwent surgery at Department of Neurosurgery of Shanghai University of Traditional Chinese Medicine (Shanghai, China). Tumor tissues were immediately frozen in liquid nitrogen and stored at −80 °C until analysis. All cases were pathologically diagnosed. Tumors were classified according to the 2007 WHO classification system .
Primary human meningeal cells were purchased from ScienCell (Carlsbad, CA, USA). IOMM-Lee and CH-157MN meningioma cell lines were obtained from the Shanghai Institute of Cell Research (Shanghai, China). Cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time PCR (qRT-PCR) analysis
Total RNA from tumor tissues and cells was extracted using TRIzol reagent (Takara, Dalian, China) following the manufacturer’s protocol. Reverse transcription was completed with random primers using the Superscript III Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on an ABI7900 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Green RT-PCR kit (QIAGEN, Valencia, CA, USA). The PCR primers are as follows : α-synuclein: forward, 5′-GCCAAGGAGGGAGTTGTGGCTGC-3′ and reverse, 5′-CTGTTGCCACACCATGCACCACTCC-3′; β-actin: forward, 5′-TCTACAATGAGCTGCGTGTG-3′ and reverse, 5′-GGTGAGGATCTTCATGAGGT-3′. The relative α-synuclein mRNA level was determined after normalization against that of β-actin.
Plasmids and transfections
Small hairpin RNA (shRNA) targeting human α-synuclein gene and negative control shRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For generation of α-synuclein-expressing plasmid, full-length human α-synuclein cDNA was amplified by PCR and cloned into pcDNA3.1(+) vector (Invitrogen). Cell transfections were performed using HiPerfect Transfection reagent (Qiagen, Hilden, Germany). After incubation for 24 h, cells were subjected to further analyses. For generation of stable clones, IOMM-Lee cells transfected with α-synuclein shRNA or control shRNA were selected with puromycin (2 μg/mL; Sigma-Aldrich) for 3 days.
BrdU incorporation assay
Cell proliferation was assessed by 5-bromo-2′-deoxyuridine (BrdU) incorporation assay using the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Applied Science, Indianapolis, IN, USA). In brief, cells were labeled using 10 μM BrdU and incubated overnight at 37 °C. The cells were then fixed and re-incubated with anti-BrdU antibody for 2 h at room temperature. The substrate solution was added and the reaction product was quantified by measuring the absorbance at 450 nm.
For analysis of apoptosis, cells were fixed in 70% ethanol and incubated with propidium iodide and Annexin-V for 30 min. Stained cells were analyzed on a flow cytometer (Becton–Dickinson Biosciences, San Jose, CA, USA).
Colony formation assay
Transfected cells were seeded onto six-well plates at a low density (600 cell per well) and cultured for 10 days. Colonies were fixed, stained with 0.1% crystal violet (Sigma-Aldrich), and counted.
In vitro wound-healing assay
Cell migration capacity was evaluated using in vitro wound-healing assay, as described previously . In brief, cells were seeded onto six-well plates and allowed to grow to confluence. The cell monolayer was scratched with a 100-μL pipette tip. After washing, the cell culture was incubated for 24 h. The percentage of wound closure was determined under a microscope. This assay was repeated three times.
Transwell invasion assay
Cells in serum-free medium (1 × 105 cells/well) were plated onto the upper chamber pre-coated with Matrigel (Becton–Dickinson Biosciences). The lower chamber was added with DMEM containing 10% FBS. After incubation for 24 h, cells that had invaded through the Matrigel were stained with 0.1% crystal violet and counted.
Eight 6–8-week-old nude mice (Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Shanghai, China) were used for tumorigenic studies. IOMM-Lee cells stably transfected with α-synuclein shRNA or control shRNA were subcutaneously injected into nude mice (n = 4; 4 × 106 cells/mouse). Tumor volume was determined every 5 days. At 25 days after cell injection, mice were sacrificed and xenograft tumors were removed and weighed.
Western blot analysis
Protein extracts from tumor tissues and cells were prepared in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Sigma-Aldrich). Protein samples were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Primary antibodies (1:500 dilution) used here are as follows: anti-Akt, anti-Akt (ser473), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-phospho-p70S6K1 (Thr389), anti-p70S6K1, anti-phospho-4EBP (Thr37/46), anti-4EBP (Cell Signaling Technology, Beverly, MA, USA), anti-α-synuclein (Abcam, Cambridge, MA, USA), and anti-β-actin (Sigma-Aldrich). Blots were developed with an enhanced chemiluminescence system (GE Healthcare Biosciences, Piscataway, NJ, USA). Densitometric analysis of the blots were performed using the Quantity One software (Bio-Rad Laboratories, Richmond, CA, USA).
Data are presented as mean ± standard deviation and were analyzed by the Student’s t test or one-way analysis of variance followed by Tukey post hoc test. The Mann–Whitney U test was used to analyze differences of α-synuclein expression in benign, atypical, and anaplastic meningiomas. P values < 0.05 were considered statistically significant.
Upregulation of α-synuclein in atypical and anaplastic meningiomas
Knockdown of α-synuclein reduces cell proliferation and colony formation and increases apoptosis in IOMM-Lee meningioma cells
Silencing of α-synuclein attenuates IOMM-Lee cell migration and invasion
The Akt/mTOR pathway is inhibited in α-synuclein-depleted IOMM-Lee cells
Overexpression of α-synuclein promotes CH-157MN cell growth and invasion
Depletion of α-synuclein retards tumor growth in a mouse xenograft model
Members of the synuclein family have been associated with the development of certain tumors . It was reported that γ-synuclein expression is dysregulated in oral squamous cell carcinoma , esophageal cancer , and breast cancer . The α-synuclein protein shows distinct tissue distributions and is predominantly expressed in brain tumors and melanomas [8, 9, 17]. In agreement with a previous study , we found that α-synuclein expression was very low in benign meningiomas. However, an increased expression of α-synuclein was observed in atypical and anaplastic meningiomas, suggesting its implication in the malignant progression of meningiomas.
To explore the biological relevance of α-synuclein upregulation, we performed loss- and gain-of-function studies. The results showed that downregulation of α-synuclein inhibited cell proliferation and colony formation in IOMM-Lee meningioma cells, whereas overexpression of α-synuclein led to opposite outcomes in CH-157MN meningioma cells. Moreover, knockdown of α-synuclein significantly triggered apoptotic death in IOMM-Lee cells, suggesting that α-synuclein is required for the growth and survival of meningioma cells. In vivo studies confirmed that α-synuclein contributes to tumorigenicity of meningioma cells. It has been documented that extracellular α-synuclein at nanomolar concentrations promotes dopaminergic neuronal survival . However, secreted α-synuclein from SH-SY5Y neuroblastoma cells exert detrimental effects on the survival of recipient neuronal cells . Another study demonstrated that overexpression of α-synuclein causes non-apoptotic death in human neuronal cells . These studies, combined with our findings, suggest that α-synuclein has distinct roles in the regulation of cell survival in different biological settings.
In addition to regulation of cell proliferation and survival, our data showed that α-synuclein also modulates the migration and invasion of meningioma cells. We observed that α-synuclein silencing suppressed the motility and invasiveness of IOMM-Lee meningioma cells. In contrast, overexpression of α-synuclein facilitated the invasion of CH-157MN meningioma cells. The pro-invasive capacity of α-synuclein provides a biological explanation for its upregulation in atypical and anaplastic meningiomas. Although there is few evidence for α-synuclein-mediated cell invasiveness, γ-synuclein has been shown to promote cell migration and invasion in different types of tumor cells such as oral squamous cell carcinoma , breast cancer , and gastric cancer .
Activation of the Akt/mTOR pathway is involved in the development and progression of meningioma [3, 4]. Fibroblast growth factor receptor-3 was found to induce the proliferation of meningioma cells via activation of the phosphoinositide 3 kinase-Akt-PRAS40-mTOR and STAT3 pathways . Pharmacological inhibition of mTOR signaling was reported to impair meningioma growth in mouse models . Inhibition of Akt activation accounts for growth reduction and apoptosis induction by depletion of astrocyte elevated gene-1 in human meningioma cells . Notably, our data showed that α-synuclein silencing significantly decreased the phosphorylation of Akt, mTOR, p70S6K, and 4EBP in IOMM-Lee cells, which provides a mechanistic explanation for the regulation of aggressive phenotypes of meningioma cells by α-synuclein.
In conclusion, α-synuclein is upregulated in atypical and anaplastic meningiomas compared to benign tumors and α-synuclein upregulation contributes to the aggressive behavior of meningioma cells via the Akt/mTOR pathway. Thus, α-synuclein represent a potential therapeutic target against malignant meningiomas.
YG and KX designed and performed the study, analyzed the data, and prepared the manuscript. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate
Written informed consent for research purposes was obtained from each patient. This study was approved by the Ethics Committee of Shanghai University of Traditional Chinese Medicine (Shanghai, China). The experiments involving animals were performed according to the institutional guidelines approved by the Shanghai University of Traditional Chinese Medicine.
This work was supported by the Scientific Research Fund from Putuo Hospital of Shanghai University of Traditional Chinese Medicine of China (2013ZD198 II).
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