Specific Expression of a New Bruton Tyrosine Kinase Isoform (p65BTK) in the Glioblastoma Gemistocytic Histotype

Abstract

Bruton's tyrosine-kinase (BTK) is a non-receptor tyrosine kinase recently associated with glioma tumorigenesis and a novel prognostic marker for poor survival in patients with glioma. The p65BTK is a novel BTK isoform involved in different pathways of drug resistance of solid tumors, thus we aimed to investigate the expression and the putative role of p65BTK in tumors of the central nervous system (CNS). We selected a large cohort of patients with glial tumors (n = 71) and analyzed the expression of p65BTK in different histotypes and correlation with clinical parameters. Sections were stained with glial fibrillary acidic protein (GFAP), p53, epidermal growth factor receptor (EGFR), S100, vimentin, and epithelial membrane antigen (EMA) antibodies. Glioma stem cell (GSC) lines, isolated from glioblastoma multiforme (GBM), were treated with different concentrations of ibrutinib, a specific inhibitor of BTK, in order to evaluate their metabolic activity, mitotic index and mortality. Moreover, an orthotopic xenotransplant of GSC from human GBM was used to evaluate the expression of p65BTK in the brain of immunodeficient mice. p65BTK was expressed in GSC and in gemistocytes in human gliomas at different histological grade. We found a significant correlation between BTK expression and low-grade (LG) tumors (p ≤ 0.05) and overall survival (OS) of patients with grade III gliomas (p ≤ 0.05), suggestive of worst prognosis. Interestingly, the expression of p65BTK remained restricted exclusively to gemistocytic cells in the xenograft mouse model. Ibrutinib administration significantly reduced metabolic activity and mitotic index and increased mortality in GSC, highlighting the specific role of p65BTK in cell proliferation and survival. In conclusion, our data demonstrated that p65BTK is expressed in glioma tumors, restricted to gemistocytic cells, has a key role in GSC and has a bad prognostic value, thus highlighting the importance of future research for targeted therapy of human gliomas.

Keywords: Bruton’s tyrosine kinase; biomarker; gemistocytes; glioblastoma; p65BTK.

Figure 1

p65BTK expression in glioma stem cell (GSC) lines (GBM2, G144, G166 and GBM04) protein extracts. Spleen protein extract was used as a control for the expression of Bruton’s tyrosine-kinase (BTK) known isoform (p77BTK). Western blots were probed with BN49 antibody for p65BTK expression, and with a commercial anti-BTK antibody (BD Transduction Laboratories) for the expression of p77BTK.

Figure 2

(A) p65BTK expression in patients with different gliomas. (B) Anatomical location and side of BTK+ tumors. (C) Positivity percentage of glial fibrillary acidic protein (GFAP), epidermal growth factor receptor (EGFR), p53 and p53-EGFR co-expression in BTK+ and BTK−. (D,F–H) BTK expression in gemistocytes in human gliomas of different grade (hGemA, human gemistocytic astrocytoma; hOD, human oligodendroglioma; haA, human anaplastic astrocytoma; hGBM, human glioblastoma multiforme; magnification 40×, scale bar: 20 μm). (E) GFAP positive gemistocytes in hGemA (magnification 60×, scale bar: 25 μm).

Figure 3

(A) Kaplan-Meier curves show significant correlation between p65BTK+ and OS in grade III gliomas. (B) No significant correlation was found with grade IV gliomas.

Figure 4

Orthotropic xenotransplant of hGBM (G144 cells) in mouse. (A) Gross anatomy of GBM in the mouse brain (magnification 2.5×, scale bar: 400 μm). (B) GFAP expression in the mouse GBM (magnification 10×, scale bar: 200 μm). (C) BTK expression in gemistocytes of xenografted human GBM tumors (magnification 40×, scale bar: 20 μm). (D) Higher magnification of gemistocytes (magnification 60×, scale bar: 10 μm).

Figure 5

Metabolic, proliferative activity and mortality of GSC lines after exposure to ibrutinib. (A–C) Dose- and time-dependent significant reduction of the metabolic activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT assay) after exposure to ibrutinib for 24, 48 or 72 h (*p ≤ 0.05; **p ≤ 0.001, treatment vs. control). (D) Reduction of mitotic index after treatment with 20 μM ibrutinib for 48 h (*p ≤ 0.05; **p ≤ 0.001, treatment vs. control). (E) Mortality essay and (F) number of live cells after exposure to ibrutinib 1 or 20 μM for 48 or 72 h (*p ≤ 0.05; **p ≤ 0.001, treatment vs. control).