MELK Inhibition Effectively Suppresses Growth of Glioblastoma and Cancer Stem-Like Cells by Blocking AKT and FOXM1 Pathways

Abstract

Glioblastoma multiforme (GBM) is a devastating disease yet no effective drug treatment has been established to date. Glioblastoma stem-like cells (GSCs) are insensitive to treatment and may be one of the reasons for the relapse of GBM. Maternal embryonic leucine zipper kinase gene (MELK) plays an important role in the malignant proliferation and the maintenance of GSC stemness properties of GBM. However, the therapeutic effect of targeted inhibition of MELK on GBM remains unclear. This study analyzed the effect of a MELK oral inhibitor, OTSSP167, on GBM proliferation and the maintenance of GSC stemness. OTSSP167 significantly inhibited cell proliferation, colony formation, invasion, and migration of GBM. OTSSP167 treatment reduced the expression of cell cycle G2/M phase-related proteins, Cyclin B1 and Cdc2, while up-regulation the expression of p21 and subsequently induced cell cycle arrest at the G2/M phase. OTSSP167 effectively prolonged the survival of tumor-bearing mice and inhibited tumor cell growth in in vivo mouse models. It also reduced protein kinase B (AKT) phosphorylation levels by OTSSP167 treatment, thereby disrupting the proliferation and invasion of GBM cells. Furthermore, OTSSP167 inhibited the proliferation, neurosphere formation and self-renewal capacity of GSCs by reducing forkhead box M1 (FOXM1) phosphorylation and transcriptional activity. Interestingly, the inhibitory effect of OTSSP167 on the proliferation of GSCs was 4-fold more effective than GBM cells. In conclusion, MELK inhibition suppresses the growth of GBM and GSCs by double-blocking AKT and FOXM1 signals. Targeted inhibition of MELK may thus be potentially used as a novel treatment for GBM.

Csnk1a1 inhibition modulates the inflammatory secretome and enhances response to radiotherapy in glioma

“…Human GBM cell lines (U87, U251, A172, T98G, LN229 and LN18) used in this study were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS). GSC2 was cultured in neurobasal medium containing basic fibroblast growth factor, epidermal growth factor, B27 supplement, harpin, l ‐glutamine and N2 supplement 26 . Primary antibodies for Csnk1a1, β‐actin, P65, p‐P65, p‐IkB and IkB were purchased from Cell Signaling Technology (CST, Beverly, MA, USA).…”

“…GSC2 cells were seeded into 96‐well plates at 1000 cells per well 26 . The cells were cultured in Neurobasal TM medium containing D4476 (2 or 4 μM), and formation of colony spheres was assessed under a microscope after 10‐14 days.…”

“…GSC2 was cultured in neurobasal medium containing basic fibroblast growth factor, epidermal growth factor, B27 supplement, harpin, l-glutamine and N2 supplement. 26 Primary antibodies for Csnk1a1, βactin, P65, p-P65, p-IkB and IkB were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). p-FOXO1a (S322/S325) and FOXO1a were purchased from Abcam (Cambridge, MA, USA).…”

“…GSC2 cells were seeded into 96-well plates at 1000 cells per well. 26 The cells were cultured in Neurobasal TM medium containing D4476…”

“…Firstly, we confirmed D4476 treatment inhibited GSC2 neurosphere formation in vitro (Figure 6A,B). Then, GCS2 cells were injected into the right hemisphere of nude mice, 26 and the sizes of the GBM xenografts were examined, as well as the survival of GBM-bearing mice. The animals were randomly divided into the control, 50 mg/kg D4476 alone, radiotherapy alone and combination (radiotherapy combined with D4476 treatment) groups (Table S3).…”