Constitutive activation of the EGFR–STAT1 axis increases proliferation of meningioma tumor cells

Abstract

Background Meningiomas are the most frequent primary brain tumors of the central nervous system. The standard of treatment is surgery and radiotherapy, but effective pharmacological options are not available yet. The well-characterized genetic background stratifies these tumors in several subgroups, thus increasing diversification. We identified epidermal growth factor receptor–signal transducer and activator of transcription 1 (EGFR–STAT1) overexpression and activation as a common identifier of these tumors. Methods We analyzed STAT1 overexpression and phosphorylation in 131 meningiomas of different grades and locations by utilizing several techniques, including Western blots, qPCR, and immunocytochemistry. We also silenced and overexpressed wild-type and mutant forms of the gene to assess its biological function and its network. Results were further validated by drug testing. Results STAT1 was found widely overexpressed in meningioma but not in the corresponding healthy controls. The protein showed constitutive phosphorylation not dependent on the JAK–STAT pathway. STAT1 knockdown resulted in a significant reduction of cellular proliferation and deactivation of AKT and ERK1/2. STAT1 is known to be activated by EGFR, so we investigated the tyrosine kinase and found that EGFR was also constitutively phosphorylated in meningioma and was responsible for the aberrant phosphorylation of STAT1. The pharmaceutical inhibition of EGFR caused a significant reduction in cellular proliferation and of overall levels of cyclin D1, pAKT, and pERK1/2. Conclusions STAT1–EGFR-dependent constitutive phosphorylation is responsible for a positive feedback loop that causes its own overexpression and consequently an increased proliferation of the tumor cells. These findings provide the rationale for further studies aiming to identify effective therapeutic options in meningioma.

Fibulin-2: A Novel Biomarker for Differentiating Grade II from Grade I Meningiomas

“…The observed overlap in grade I and II plasma Fibulin-2 levels was not surprising. Previous studies have described intra-tumoural biological heterogeneity in meningiomas [ 9 , 31 , 32 ] and a spectrum of biological behaviour in the grade II tumours [ 9 , 10 , 11 , 13 ], with biomarker validation studies often showing variable protein expression in these tumours, and similar expression profiles when compared to grade I and III meningiomas [ 12 , 33 ].…”

GATA-4, a potential novel therapeutic target for high-grade meningioma, regulates miR-497, a potential novel circulating biomarker for high-grade meningioma

“…Human meningeal cells (HMC, Cat# 1400) were obtained from Sciencell™ and maintained following the manufacturer's protocol. The malignant meningioma cell line KT21-MG1-Luc5D (RRID:CVCL_JK00), the benign meningioma cell line Ben-Men-1 (RRID:CVCL_1959), and WHO I primary meningioma cells (PDMN) were maintained as previously described [36] , [37] , [38] . Briefly, tumour samples were washed twice with sterile PBS (Gibco, Life Technologies, Loughborough, UK), and transferred into a P100 sterile plate, where they were minced into small pieces and dissociated in DMEM (Gibco, Life Technologies, Loughborough, UK), with 10% FBS (Sigma Aldrich, Gillingham, UK), 100 U/mL penicillin/streptomycin, and 20 u/mL Collagenase III (Worthington Biomedical Corp., Lakewood, NJ) overnight at 37 °C.…”

“…WHO II meningioma primary cells were isolated from resected tumours following the same protocol as WHO I PDMN cells, but were maintained in Dulbecco's Modified Eagle Medium F-12 Nutrient Mixture (Ham) (DMEM/F-12 (1:1)(1X) + GlutaMAX™-I; Thermo Fisher Scientific, Loughborough, UK), supplemented with 20% FBS (Sigma Aldrich, Gillingham, UK), 1% d -(+)-glucose (Sigma Aldrich, Gillingham, UK), and 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific, Loughborough, UK). All PDMN cells were used between passage 3 and 5, and were tested for Epithelial Membrane Antigen (EMA) and Vimentin expression via immunocytochemistry [ 37 , 38 ].…”

miR-9-1 Is a New Circulating Biomarker for Higher-grade Meningioma Regulated via the EGFR-FOS Network

“…To demonstrate that EGFR activation leads to an increase of FOS, which results in increased miR-9-1, we stimulated KT21-MG1 and IOMM-Lee cells with EGF for 30 and 120 minutes (Fig. 3a) (22). Activation of the EGF receptor determined an increase in FOS levels (1.97 and 1.81 folds at 120 minutes, when compared to unstimulated KT21-MG1 and IOMM-Lee cells, respectively), and a signi cant increase of miR-9-1 ( Fig.…”

“…The benign meningioma cell line Ben Men-1 (RRID:CVCL_1959), and the malignant meningioma cell line IOMM-Lee (RRID:CVCL_5779) were both maintained as in (21). WHO I primary meningioma cells were obtained as reported in (22) Gene expression levels were computed using the quantitative 2 -(ΔΔCt) method, employing the HMC as calibrator, in triplicate (24).…”